Viral gene therapy as treatment for cholesterol storage disease or disorder

ABSTRACT

Provided herein are compositions and methods for the viral gene therapy (e.g., AAV-directed gene therapy) of cholesterol storage diseases or disorders, such as Niemann-Pick disease, Type C.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/144,702, filed Apr. 8, 2015, and entitled VIRALGENE THERAPY AS TREATMENT FOR CHOLESTEROL STORAGE DISEASE OR DISORDER,which is incorporated herein by reference in its entirety.

GOVERNMENT FUNDING

Research supporting this application was carried out by the UnitedStates of America as represented by the Secretary, Department of Healthand Human Services.

BACKGROUND OF THE INVENTION

Niemann-Pick disease, type C (NPC) is a rare and fatal, autosomalrecessive, neurodegenerative disease that can present in infants,children, or adults. Its incidence in persons of Western Europeandescent is 1/90,000 (Wassif C A et al., “High incidence of unrecognizedvisceral/neurological late-onset Niemann-Pick disease, type C1,predicted by analysis of massively parallel sequencing data sets,” GenetMed. 2015 Mar. 12). Approximately 95% of patients with NPC havemutations in NPC1, a gene implicated in intracellular cholesteroltrafficking. Mutation of NPC1 causes intracellular accumulation ofunesterified cholesterol in late endosomal/lysosomal structures andmarked accumulation of glycosphingolipids, especially in neuronaltissue. Thus, NPC patients generally present with hepatosplenomegaly(enlargement of liver and spleen) and neurological degeneration.

A prenatal syndrome of nonimmune fetal hydrops can be the first symptomof NPC disease. Neonates can present with severe liver disease frominfiltration of the liver and/or respiratory failure. Other infants,without liver or pulmonary disease, have hypotonia and developmentaldelay. The classic presentation occurs in mid-to-late childhood with theinsidious onset of ataxia, vertical supranuclear gaze palsy (VSGP), anddementia. Regression is common. Seizures are frequent and neurologicalsymptoms become disabling, making oral feeding impossible; death usuallyoccurs in the late second or third decade from aspiration pneumonia.Adults can be more mildly affected and are more likely to present withdementia or psychiatric symptoms. There are no proven treatments forNPC, and after the diagnosis, fatal neurodegeneration is inevitable. Thefact that most patients have disease onset in childhood makes the searchfor effective therapies urgent.

The diagnosis of NPC disease is confirmed by specialized biochemicaltesting that demonstrates cholesterol storage and is detected by filipinstaining in cultured fibroblasts. Most individuals with NPC disease haveNPC type 1, caused by mutations in NPC1; fewer than 20 individuals havebeen diagnosed with NPC type 2, caused by mutations in NPC2. Moleculargenetic testing of NPC1 and NPC2 detects disease-causing mutations inapproximately 94% of individuals with NPC disease, almost all of whomhave mutations in NPC1. NPC disease, regardless of the locus andallele(s), is a recessive metabolic condition and the mutations are lossof function or reduced function. Therefore providing and expressing asingle copy of the wild type gene can completely restore NPC1 or 2enzymatic function.

A series of landmark studies conducted by the research group of Dr.William Pavan of the NHGRI/NIH led to the identification of both themouse and human genes for NPC1 (Loftus et al. Science 277: 232-35;Carstea et al. Science 277: 228-31). A murine model of NPC, Npc^(nih)(also called BALB/cNctr-Npc1^(mIN)/J), arising from a spontaneousframe-shift mutation in the Npc1 gene has been described and extensivelycharacterized during these research efforts (Loftus et al. Science 277:232-35). Npc^(nih) homozygotes have an early, severe, and rapidlyprogressing disease, which is characterized by weight loss, ataxia, andlethality by 9 weeks of age. The mutation carried by this mouse is anull, and Npc^(nih) homozygous mice fail to make Npc1 protein or mRNA.This animal model also displays neurological symptoms and earlylethality: Npc^(nih) homozygous mice uniformly begin losing weight by 6weeks of age and do not survive past 9 weeks. Thus, these animalsrepresent an ideal model of human NPC disease caused by loss of functionmutations in the gene NPC1.

Over the years, other mouse models of NPC disease, specifically causedby varied natural or engineered mutations in the mouse Npc1 gene, havebeen generated but display less severe of a disease phenotype. All mousemodels of NPC disease caused by mutation or other malfunction of theNpc1 gene in any mouse strain are treatable by the vector andderivatives described herein and are encompassed in said claims. Suchmodels, as a group including Npc^(nih) homozygous animals, are generallyconsidered Npc^(−/−) designating homozygous Npc loss-of-functionalleles, of which Npc^(nih) is paradigmatic.

Notwithstanding the development of such mouse models, no curativetherapy for NPC yet exists. A strategy or methodology for clinicallytreating NPC and/or providing a curative therapy for NPC and/or itssymptoms is urgently needed in the art. The present invention fulfillssuch a need.

SUMMARY OF THE INVENTION

The present invention provides, for the first time, gene therapyconstructs comprising a therapeutic human nucleic acid molecule which isable to correct the cellular defect characteristic of certaincholesterol storage diseases or disorders, such as, Niemann-Pick type Cdisease (“NPC”), when the therapeutic human nucleic acid molecule isunder control of a tissue-specific promoter, including, in certainembodiments, a neuronal-specific calcium/calmodulin-dependent proteinkinase II (CaMKII) promoter or one broadly expressed, such as theelongation factor 1 alpha (EF1α) constitutive promoter or a derivativethereof. The inventors contemplate use of these vector constructs forgene therapy of certain cholesterol storage diseases or disorders,including NPC. Examples are provided which demonstrate the reduction ofpractice and the effectiveness of the present invention in the mostestablished and well-characterized animal model of NPC.

More in particular, the invention provides compositions and methods fortreating or preventing cholesterol storage diseases or disorders. Incertain aspects, the present invention provides compositions and methodsfor treating or preventing Niemann-Pick disease, type C. In certainembodiments, the invention relates to compositions and methods fortreating or preventing cholesterol storage diseases or disorders thatare characterized by or associated with a risk of diminution of centralnervous system (CNS) function, including NPC. In still otherembodiments, the present invention relates to nucleic acid moleculesencoding therapeutic transgenes, e.g., NPC1 or NPC2, which are capableof restoring the function lost to one or more defective genes orpolypeptide products thereof, e.g., a mutant NPC1 or NPC2 gene. In yetother embodiments, the invention relates to pharmaceutical compositionsthat are suitable for administering therapeutically effective amountsthe nucleic acid molecules of the invention. In still furtherembodiments, the present invention relates to methods for diagnosing NPCand/or monitoring the progress of gene therapy treatment of NPC bymonitoring the expression and/or function of a therapeutic gene, e.g.,NPC1 or NPC2.

The present invention, in still other embodiments, relates to methods ofgene therapy involving administering in an effective amount a nucleicacid molecule comprising a therapeutic transgene, e.g., NPC1 or NPC2, inorder to treat or prevent a cholesterol storage disease or disorder,including NPC. In still other embodiments, the present invention relatesto methods of gene therapy involving administering, in an effectiveamount, an expression vector encoding NPC1 or NPC2 in order to treat orprevent a cholesterol storage disease or disorder, including NPCdisease. In yet other embodiments, the nucleic acid molecule and/orexpression vector may be selectively delivered to a target site ortissue, e.g., the central nervous system.

The nucleic acid molecules or gene therapy constructs in certainembodiments comprising one or more therapeutic transgenes, e.g., NPC1 orNPC2, which are under the control of at least one genetic regulatoryelement, such as a promoter. In certain embodiments, the promoter is atissue-specific promoter that is capable of being expressed in the CNS.In some embodiments, the promoter is a neuronal-specific calmodulinpromoter. In other embodiments, the promoter is an EF1α (“humanelongation factor 1 alpha”) constitutive promoter. In other embodimentsthe promoter is a novel, truncated variant of the EF1α (_(miniEF1α)).

The present invention also relates to specific nucleic acid moleculescomprising a therapeutic transgene, e.g., NPC1 or NPC2, undertranscriptional control of a promoter that is capable of being expressedin the CNS, including a neuronal-specific calmodulin promoter or a_(miniEF1α) constitutive promoter. The invention also contemplates thatsuch nucleic acid constructs may be engineered into any suitable genetherapy vector, such as a retrovirus, lentivirus adenovirus oradeno-associated virus (AAV) vector, nucleic acid such as plasmid DNA,peptide nucleic acids, or mRNA, including mRNAs that are containmodified bases to enhance in vivo expression. All forms of nucleic acidscan be delivered without further modification, such as naked DNA, orpackaged into nanoparticles or lipid nanoparticles and delivered in anappropriate fashion to produce NPC1 or 2 expression. In a particularembodiment, the background gene therapy vector is an AAV.

Through the manipulation of the nucleotide sequences provided by thisinvention by standard molecular biology techniques, variants of the NPC1and NPC2 proteins may be made which differ in precise amino acidsequence from the disclosed proteins yet which maintain the basicfunctional characteristics of the disclosed NPC1 and NPC2 proteins orwhich are selected to differ in some characteristics from theseproteins. Such variants are another aspect of the present invention asthey may also be administered using the gene therapy vectors andtissue-specific promoters (e.g., neuronal-specific CaMKII or_(mini)CaMKII promoter) or constitutive promoters (e.g., EF1α or_(miniEF1α)) of the invention.

In another embodiment, the vectors described here may be modified toencode versions of the NPC1 and NPC2 proteins that have beencodon-optimized for expression in a test subject, such as a mouse or cator human, or for use in patients with NPC disease.

The foregoing and other features and advantages of the invention willbecome more apparent from the following detailed description andaccompanying drawings. Those skilled in the art will appreciate that theutility of this invention is not limited to the specific experimentalmodes and materials described herein.

In a preferred embodiment, the present invention is directed to anucleic acid construct comprising: (1) a viral vector sequence; and (2)an NPC1 gene sequence under control of a mini-calmodulin promoter or amini-elongation factor 1 α (_(shortminiEF1α)) promoter.

The viral vector can be an adeno-associated viral (AAV) vector.

The nucleic acid construct can comprise a sequence selected from thegroup consisting of SEQ ID NO: 1 and SEQ ID NO: 7.

The nucleic acid construct can be used to treat or prevent a cholesterolstorage disease or disorder in a subject, said method comprising:administering a nucleic acid construct of any one of the precedingclaims and a pharmaceutically acceptable viral carrier to a subject,thereby treating or preventing the cholesterol storage disease ordisorder in said subject.

The cholesterol storage disease or disorder can be Niemann-Pick disease,Type C.

The subject can be a mouse or other animal, e.g., an experimentalanimal. The animal can be a Npc1 knockout mouse.

The subject can also be a human, who has or is at risk of having NPC.

The nucleic acid construct can be encapsidated with an AAV serotype 9capsid.

The concentration of the nucleic acid construct in the viralcarrier-nucleic acid construct composition can be 5×10¹² gc/ml orgreater (“genome copy” per ml).

The pharmaceutically acceptable viral carrier can be AAV.

The AAV can be selected from the group consisting of AAV2, AAV3, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh8, AAVrh10, AAVrh33, AAV rh34, AAVAnc80, or AAV PHP.B.

The pharmaceutically acceptable viral carrier can comprise a viralcapsid selected from the group consisting of AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV rh8, AAVrh10, AAVrh33, AAV rh34, AAV Anc80or AAV PHP.B viral capsid.

In another aspect, the invention relates to a method for treatingNeimann-Pick disease. Type C in a subject by gene therapy comprisingadministering a composition comprising a therapeutically effectiveamount of gene therapy construct comprising (1) a viral vector sequence;and (2) an NPC1 gene sequence under control of a mini-calmodulinpromoter or a mini-elongation factor 1 α (_(mini)EF1α) promoter, and apharmaceutically acceptable carrier.

The viral vector can be an adeno-associated viral (AAV) vector.

The gene therapy construct can comprise a sequence selected from thegroup consisting of SEQ ID NO: 1 (AAV._(mini)CaMKII NPC1.RBG) and SEQ IDNO: 7 (pAAV-_(mini)EF1α-NPC1-RBG).

The subject can be a mouse or other animal, e.g., an experimentalanimal. The animal can be an Npc1 knockout mouse.

The subject can also be a human, who has or is at risk of having NPC.

The gene therapy construct can be encapsidated with an AAV serotype 9capsid.

The composition can comprise the gene therapy construct at aconcentration of 5×10¹² gc/ml or greater.

The viral vector sequence can be AAV, which can be selected from thegroup consisting of AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh33, AAV rh34, AAV Anc80 or AAV PHP.B.

The gene therapy construct can comprise a viral capsid selected from thegroup consisting of AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh33, AAV rh34, AAV Anc80 or AAV PHP.B viral capsid.

Other aspects of the invention are described in, or are obvious from,the following disclosure, and are within the ambit of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings. Reference will now be made, by way of example, to theaccompanying drawings which show example embodiments of the presentapplication.

FIG. 1 shows a map of the _(mini)calmodulin-NPC plasmid(pAAV._(mini)CaMKII NPC1.RBG) as disclosed herein for AAV-mediateddelivery in AAV-_(mini)calmodulin-NPC.

FIG. 2 shows the nucleotide sequence of AAV._(mini)CaMKII NPC1.RBG (SEQID NO: 1).

FIG. 3 shows the nucleotide sequence of the 5′ inverted terminal repeat(5′ ITR) of AAV._(mini)CaMKII NPC1.RBG (SEQ ID NO: 2).

FIG. 4 shows the nucleotide sequence of the CaMKII promoter(_(mini)calmodulin promoter) of AAV._(mini)CaMKII NPC1.RBG (SEQ ID NO:3).

FIG. 5 shows the nucleotide sequence of the hNPC1 cDNA ofAAV._(mini)CaMKII NPC1.RBG (SEQ ID NO: 4).

FIG. 6 shows the nucleotide sequence of the rabbit globin polyA ofAAV._(mini)CaMKII NPC1.RBG (SEQ ID NO: 5).

FIG. 7 shows the nucleotide sequence of the 3′ inverted terminal repeat(3′ ITR) of AAV._(mini)CaMKII NPC1.RBG (SEQ ID NO: 6).

FIG. 8 shows survival in Npc^(nih) homozygous mice (n=10) after notreatment (black hatched lines), treatment with a AAV2/9._(mini)CaMKIIeGFP.RBG reporter vector (n=6) (green line) or treatment withAAV2/9._(mini)CaMKII NPC1.RBG (n=9)(blue line).

FIG. 9. Map of AAV-_(mini)EF1α-NPC1 showing important vector elements.The full length EF1α promoter was truncated (orange) and tested for theability to drive eGFP expression in a transfection experiment using 293Tcells (not presented). The promoter fragment was then introduced into anAAV vector in front of the full length human NPC1 cDNA (blue), followeda rabbit beta globin poly A signal, and flanked by AAV2 invertedterminal repeats (ITR), shown in yellow. The resulting vector waspackaged into an AAV using a serotype 9 capsid to createAAV2/9-_(mini)EF1α-NPC1 or AAV9-_(mini)EF1α-NPC1.

FIG. 10 shows the nucleotide sequence of AAV-_(mini)EF1α-NPC. (SEQ IDNO: 7)

FIG. 11 shows the nucleotide sequence of the _(mini)EF1α promoter ofAAV2/9-_(mini)EF1α-NPC. (SEQ ID NO: 8)

FIG. 12 GenBank Accession No. BC063302 (Homo sapiens Niemann-Pickdisease, type C1, mRNA (cDNA clone), which provides the NPC1 polypeptidesequence. (SEQ ID NO: 9).

FIG. 13 GenBank Accession No. BC063302 (Homo sapiens Niemann-Pickdisease, type C1, mRNA (cDNA clone), which provides the NPC1 cDNA codingsequence. (SEQ ID NO: 10).

FIG. 14 GenBank Accession No. BC002532 (Homo sapiens Niemann-Pickdisease, type C2, mRNA (cDNA clone), which provides the NPC2 polypeptideamino acid sequence (SEQ ID NO: 11).

FIG. 15 GenBank Accession No. BC002532 (Homo sapiens Niemann-Pickdisease, type C2, mRNA (cDNA clone), which provides the NPC2 cDNAnucleotide sequence (SEQ ID NO: 12).

FIG. 16 (A-G) show neuronal distribution of GFP in the Npc1^(−/−) mousebrain after retro-orbital injection of AAV2/9-_(mini)CaMKII-GFP. (a)Excerpt from the Allen Brain Atlas, demonstrating the neuronalexpression pattern of CaMKII in the wild type mouse brain. (b)Immunofluorescence of AAV9-_(mini)CaMKII-GFP (green) in the Npc1^(−/−)brain after retro-orbital injection. (c-g) Co-localization of the GFPsignal with NeuN immunofluorescence (red), indicating incorporation ofAAV2/9-_(mini)CaMKII-GFP into neuronal populations, including corticalpyramidal neurons (d, green removed in e to show double labeling,arrowheads) and CA3 hippocampal neurons (f, green removed in g to showdouble labeling, arrowheads).

FIG. 17 (A-D) shows survival of Npc1^(−/−) mice and growth followingAAV9 treatments. (a) Kaplan-Meier Curve depicts survival of: Npc1^(−/−)pups (n=6) treated with 2×10¹¹ GC of AAV2/9-_(mini)CaMKII-NPC1 between 1and 3 days, Npc1^(−/−) mice (n=9) treated with 1×10¹² GC ofAAV2/9-_(mini)CaMKII-NPC1 between 20 and 25 days of life, Npc1^(−/−)mice (n=6) treated with 1×10¹² GC of AAV2/9-_(mini)CaMKII-GFP between 20and 25 days of life and untreated Npc1^(−/−) mice (n=16). (b) Survivaldata depicted as a vertical scatter plot to show survival distribution.(c) Week at which Npc1^(−/−) mice reached peak weight. (d) Percentageweight change between weeks 6 and 9. ** P<0.01, ***P<0.001, Log-ranked(Mantel Cox) test or t-test two-tailed.

FIG. 18 (A-V) shows the effect of AAV2/9-_(mini)CaMKII-NPC1 treatment inthe CA3 hippocampus and layer V neocortex of Npc1^(−/−) mice. (a-r)Immunohistochemical imaging of Npc1 or NPC1 protein levels (red) in thehippocampus and layer V neocortex, co-stained with NeuN (green) andfilipin (blue). (a-f) Endogenous Npc1 expression in the Npc1^(+/+)mouse, with NeuN stain removed in (b, d, f) to better show the neuronalNpc1 or NPC1 expression and magnified images of layer V neocortex (c-d)and CA3 hippocampus (e-f). (g-l) Endogenous levels of Npc1 or NPC1protein in the Npc1^(−/−) mouse, with NeuN stain removed in (h, j, l) tobetter show the lack of Npc1 or NPC1 expression and high level ofintracellular filipin inclusions, with magnified images of layer Vneocortex (c-d) and CA3 hippocampus (e-f). (m-r) NPC1 protein levels inthe Npc1^(−/−) mice injected with AAV2/9-_(mini)CaMKII-NPC1. NeuN stainremoved in (n, p, r) to better show the presence of NPC1 expression insome neurons and the reduced level of intracellular filipin inclusions,with magnified images of layer V neocortex (o-p), and CA3 hippocampus(q-r). Quantification of filipin and Npc1 or NPC1 mean pixel intensityof the neuronal cell body in layer V neocortical neurons (s-t) and CA3hippocampal neurons (u-v), data expressed as mean±S.E.M. A.U.=arbitraryunits. * p<0.05, ** p<0.01, **** p<0.0001, one-way ANOVA with Tukey'spost-test.

FIG. 19 (A-N) shows biochemical correction of the cholesterol storagephenotype in neurons transduced with AAV9-_(mini)CaMKII-NPC1 in theNpc1^(−/−) mouse. Immunohistochemical imaging of NPC1 protein levels(red) in the hippocampus (a-b) and layer V neocortex (e-f), co-stainedwith filipin (blue) and NeuN (green, in a and e only). Arrows indicateneurons without appreciable Npc1 protein and high filipin staining.Arrowheads indicate neurons successfully infected by theAAV9-_(mini)CaMKII-NPC1 with strong NPC1 staining and reduced filipinlabeling. (c-d) NPC1 intensity of all layer V neurons measured, plottedagainst filipin intensity. (g-h) NPC1 intensity of all CA3 hippocampalneurons measured, plotted against filipin intensity. Upper leftquadrants in (d, h) indicate the percentage of Npc1^(−/−) neuronscorrected to control levels with AAV9-_(mini)CaMKII-NPC1 treatment.Imaged density of AAV-_(mini)CaMKII-GFP (i-j) andAAV9-_(mini)CaMKII-NPC1 (k-l) incorporation in the layer V cortex (i-k)and CA3 hippocampus (j-l), with quantification in (m-n).ns=non-significant.

FIG. 20 (A-F) shows delayed Purkinje cell death afterAAV2/9-_(mini)CaMKII-NPC1 treatment in Npc1^(−/−) mice.Immunofluorescent calbindin staining of Purkinje cells (green) inNpc1^(−/−) (a), Npc1^(−/−) (b) and Npc1^(−/−) (c) mice at 9 weeks of age(I-X=cerebellar lobular i.d. in c). (d-f) Quantification of Purkinjecell number in posterior cerebellar lobules. All data in bar-graphsexpressed as mean±S.E.M., * p<0.05, ** p<0.01, one-way ANOVA withTukey's post-test.

FIG. 21. Effect of AAV2/9-_(mini)EF1α-NPC1 treatment on Npc^(−/−) mouseweight. Npc1^(−/−) mice were injected retro-orbitally at p24 with1.21e12 GC of AAV9-EF1a-NPC1. Survival and weight gain have beenserially monitored since injection. Note that some of the treatedmutants achieved weight equal to that of wild type, unaffectedlittermates and remarkably, have more than doubled survival compared toNpc1^(−/−) controls (see FIG. 2a and FIG. 9). The cohort of mice in thispilot study are alive at the time of this PCT update. A comparison to*untreated and **AAV9-_(mini)CaMKII-NPC1 treated Npc1^(−/−) mice isindicated at the bottom of the figure.

FIG. 22(A-B) show weight effect of AAV2/9-_(mini)EF1α-NPC1 vsAAV2/9-_(mini)CaMKII-NPC1 treatment. Untreated or AAV treated Npc1^(−/−)mice were serially weighed and compared to previous weights (a) or theage of peak weight; *p<0.01 *** p<0.001 compared to untreated. (b). Micetreated with AAV2/9-_(mini)EF1α-NPC1 achieved a peak weight later thanthe other groups, showing the AAV gene therapy allows the mice tocontinue to gain weight much longer than untreated mice or those thatreceived the AAV2/9-_(mini)CaMKII-NPC1 vector; *p<0.01.

FIG. 23 shows survival differences between AAV2/9-_(mini)EF1α-NPC1 vsAAV2/9-_(mini)CaMKII-NPC1. Kaplan-Meier curve depicts survival of:untreated Npc1^(−/−) mice (n=16), Npc1^(−/−) mice (n=9) treated with1×10¹² GC of AAV9-_(mini)CaMKII-NPC1 between 20 and 25 days of life, andNpc1^(−/−) mice (n=7) treated with 1×10¹² GC of AAV9-_(mini)EF1α-NPC1between 20 and 25 days of life. *** p<0.001 compared toAAV9-_(mini)CaMKII-NPC1 treated Npc1^(−/−) mice.

SEQUENCE LISTING

The specification includes a Sequence Listing appended herewith whichincludes sequences, as follows:

SEQ ID NO: 1: Nucleotide sequence of AAV._(mini)CaMKII NPC1.RBG (FIG.2);

SEQ ID NO: 2: Nucleotide sequence of the 5′ inverted terminal repeat (5′ITR) of AAV._(mini)CaMKII NPC1.RBG (FIG. 3);

SEQ ID NO: 3: Nucleotide sequence of the CaMKII promoter(_(mini)calmodulin promoter) of AAV._(mini)CaMKII NPC1.RBG (FIG. 4);

SEQ ID NO: 4: Nucleotide sequence of the hNPC1 cDNA of AAV._(mini)CaMKIINPC1.RBG (FIG. 5);

SEQ ID NO: 5: Nucleotide sequence of the rabbit globin polyA ofAAV._(mini)CaMKII NPC1.RBG (FIG. 6);

SEQ ID NO: 6: Nucleotide sequence of the 3′ inverted terminal repeat (3′ITR) of AAV._(mini)CaMKII NPC1.RBG (FIG. 7);

SEQ ID NO: 7: Nucleotide sequence of AAV_(mini)EF1α-NPC (FIG. 10);

SEQ ID NO: 8: Nucleotide sequence of the EF1α promoter ofAAV2/9-_(mini)EF1α-NPC (FIG. 11);

SEQ ID NO: 9: NPC1 amino acid sequence (FIG. 12);

SEQ ID NO: 10: NPC1 cDNA nucleotide sequence (FIG. 13);

SEQ ID NO: 11: NPC2 amino acid sequence (FIG. 14); and

SEQ ID NO: 12: NPC2 cDNA nucleotide sequence (FIG. 15).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed, at least in part, to compositions andmethods for treating or preventing cholesterol storage diseases ordisorders, such as Niemann-Pick disease, type C caused by mutation ormalfunction of the NPC1 and/or NPC2 enzymes, which are encoded by theNPC1 and NPC2 genes, respectively. In certain aspects, compositions ofthe instant invention include one or more gene therapy constructscomprising NPC1 and/or NPC2 genes, or derivatives and/or mutantsthereof, which are operably linked to at least a promoter element thatis capable of being expressed in a tissue of the central nervous system.In certain embodiments, the promoter is a neuronal-specific calmodulinpromoter. In other embodiments, the promoter is a constitutive promoter,e.g., an EF1α or _(mini)EF1α constitutive promoter, which is capable ofbeing expressed in neuronal as well as other tissues. As demonstrated bya reduction to practice using accepted NPC mouse models, the genetherapy vectors of the present invention were effective in treatingand/or preventing NPC.

In order to facilitate review of the various embodiments of theinvention, the following definitions of terms and explanations ofabbreviations are provided, as follows:

Definitions

The instant invention provides for the therapeutic or prophylactic useof gene therapy vectors to achieve treatment of subjects having or atrisk of developing a cholesterol storage disease or disorder. In certainembodiments, the invention provides compositions and methods fortreating or preventing Niemann-Pick disease, type C1, either by deliveryof the vector to the CNS in a targeted manner, or systemically, usingrecombinant AAV viral vectors (e.g., AAV9 viral vectors) to achieveeffective transgene delivery in a subject and/or the cells of a subject.In related embodiments, the transgene is NPC1 or functional variant orfragment thereof. In other embodiments, the transgene is NPC2 orfunctional variant or fragment thereof.

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

General Terms

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude other elements. “Consisting essentially of”, when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination. Thus, a compositionconsisting essentially of the elements as defined herein would notexclude trace contaminants from the isolation and purification methodand pharmaceutically acceptable carriers, such as phosphate bufferedsaline, preservatives, and the like. “Consisting of” shall meanexcluding more than trace elements of other ingredients and substantialmethod steps for administering the compositions of this invention.Embodiments defined by each of these transition terms are within thescope of this invention.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

The term “obtaining” is understood herein as manufacturing, purchasing,or otherwise coming into possession of.

As used herein, “kits” are understood to contain at least thenon-standard laboratory reagents of the invention and one or morenon-standard laboratory reagents for use in the methods of theinvention.

The terms “about” and “approximately” shall generally mean an acceptabledegree of error for the quantity measured given the nature or precisionof the measurements. Typically, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values. Alternatively, and particularly inbiological systems, the terms “about” and “approximately” may meanvalues that are within an order of magnitude, preferably within 10- or5-fold, and more preferably within 2-fold of a given value. Numericalquantities given herein are approximate unless stated otherwise, meaningthat the term “about” or “approximately” can be inferred when notexpressly stated.

Terms Relating to NPC

The term Niemann-Pick disease, Type C or abbreviated as “NPC,” refers tothe disorder as it is known in the medical art, and is distinct fromType A or B. NPC patients are not able to metabolize cholesterol andother lipids properly within the cell. Consequently, excessive amountsof cholesterol accumulate within the liver and spleen and excessiveamounts of other lipids accumulate in the brain. NPC causes a secondaryreduction of ASM (acid sphingomyelinase) activity such as ischaracteristic of Type A and B. Type C Niemann-Pick disease has anestimated 500 cases diagnosed worldwide. It is believed, however, thatthe number of people affected by NPC is higher, but diagnosticdifficulties do not allow an accurate assessment of the occurrence rate.NPC has been initially diagnosed as a learning disability, mildretardation, “clumsiness,” and delayed development of fine motor skills.It is not uncommon for a family to spend several years seeking adiagnosis before NPC is identified. NPC is always fatal. The majority ofchildren with NPC die before age 20 (many die before the age of 10).Late onset of symptoms can lead to longer life spans but it is extremelyrare for any person with NPC to reach age 40. A recent study based ongenomic analyses suggests the incidence of infantile onset NPC is1:90,000 but when all forms are considered, including the adult onsetvariants, the disease may be as common as 1/19,000-1/36,00. There iscurrently no curative therapy for any form of NPC disease.

The term “NPC1” refers to the wildtype NPC1 gene or protein, variousmutant forms of which are associated with Neimann-Pick Type C disease byleading to the accumulation of intracellular unesterified cholesterol.For convenience, the human gene is referred to as hNPC1 or NPC1 and themurine gene as mNPC1 or Npc1 (this same nomenclature is also used todistinguish between the human and murine cDNAs and proteins). Where no“h” or “m” designation is given, reference to the human NPC1 genegenerally is intended. The definition of an NPC1 gene includes thevarious sequence polymorphisms that exist in the species in question,i.e., the term “hNPC1” or a wildtype hNPC1 encompasses all varioussequence polymorphisms in humans.

The NPC1 protein or a derivative may be functionally characterized byits ability, when expressed in NPC cells, to correct the lysosomalcholesterol accumulation phenotype that is characteristic of such cells.Thus, “NPC1 protein biological activity” refers to the ability of aprotein to correct the lysosomal cholesterol accumulation phenotype thatis characteristic of NPC cells.

A “wildtype NPC1 protein” refers to any protein encoded by a wild-typegene that is capable of having normal (level of function absent diseaseor disorder) biological activity when expressed or introduced in vivo.Such functionality can be tested by any means known to establishfunctionality of a protein.

The term “NPC1 derivative gene.” which can include a “mutant NPC1 gene,”refers to any non-wildtype NPC1 sequence. Typically, a “mutant NPC1gene” refers to a non-wildtype sequence that results in an abberantfunctioning NPC1 protein, and thus, NPC disease. However, the term “NPC1derivative gene” is meant to be broad enough to encompass an NPC1 mutantgene, but also any other NPC1 gene carrying a genetic change that mayresult an NPC1 protein having any of an increase, a decrease, or nochange in activity as compared to the wildtype protein.

The term “NPC1 protein, derivative, or functional variant thereof.”which can include a “mutant NPC1 protein,” refers to any non-wildtypeNPC1 sequence or fragment thereof. Typically, a “mutant NPC1 protein”refers to a non-wildtype NPC1 polypeptide that has an abberant functionas compared to a wildtype NPC1 protein, and which results in NPC1disease. However, the term “NPC1 protein, derivative, or functionalvariant thereof” is meant to be broad enough to encompass an NPC1 mutantprotein, but also any other NPC1 protein carrying a genetic change(including a fragment) that may result an NPC1 protein having any of anincrease, a decrease, or no change in activity as compared to thewildtype NPC1 protein. In the case of the present invention, the “NPC1protein, derivative, or functional variant thereof” can also refer tohomologous NPC1 proteins from non-human sources, e.g., mouse, monkey,horse, rabbit, and the like.

The term “NPC2” refers to the wildtype NPC1 gene, various mutant formsof which are associated with Neimann-Pick Type C disease by leading tothe accumulation of intracellular unesterified cholesterol. Forconvenience, the human gene is referred to as hNPC2 or NPC2 and themurine gene as m NPC2 or Npc2 (this same nomenclature is also used todistinguish between the human and murine cDNAs and proteins). Where no“h” or “m” designation is given, reference to the human NPC2 genegenerally is intended. The definition of an NPC2 gene includes thevarious sequence polymorphisms that exist in the species in question,i.e., the term “hNPC2” or a wildtype hNPC2 encompasses all varioussequence polymorphisms in humans.

The term “NPC2 derivative gene,” which can include a “mutant NPC2 gene,”refers to any non-wildtype NPC2 sequence. Typically, a “mutant NPC2gene” refers to a non-wildtype sequence that results in an abberantfunctioning NPC2 protein, and thus, NPC disease. However, the term “NPC2derivative gene” is meant to be broad enough to encompass an NPC2 mutantgene, but also any other NPC2 gene carrying a genetic change that mayresult an NPC2 protein having any of an increase, a decrease, or nochange in activity as compared to the wildtype protein.

The term “NPC2 protein, derivative, or functional variant thereof,”which can include a “mutant NPC2 protein,” refers to any non-wildtypeNPC2 sequence or fragment thereof. Typically, a “mutant NPC2 protein”refers to a non-wildtype NPC2 polypeptide that results has an abberantfunction as compared to a wildtype NPC1 protein, and which results inNPC2 disease. However, the term “NPC2 protein, derivative, or functionalvariant thereof” is meant to be broad enough to encompass an NPC2 mutantprotein, but also any other NPC2 protein carrying a genetic change(including a fragment) that may result an NPC2 protein having any of anincrease, a decrease, or no change in activity as compared to thewildtype NPC2 protein. In the case of the present invention, the “NPC2protein, derivative, or functional variant thereof” can also refer tohomologous NPC2 proteins from non-human sources, e.g., mouse, monkey,horse, rabbit, and the like.

The term “NPC sufferer” or “NPC homozygote” refers to a person whocarries a mutant NPC1 or NPC2 gene, such that the person exhibitsclinical symptoms of Niemann-Pick type C disease.

The term “NPC carrier” or “NPC heterozygote” refers to a person who doesnot exhibit clinical symptoms of NPC but who carries one mutant form ofthe NPC1 or NPC2 gene and may transmit this mutant gene to progeny.

As used herein, the term “cholesterol storage disease or disorder” ismeant to refer to a disease or disorder of or related to cholesterolmetabolism, optionally that is treatable via use of gene therapy fordelivery of NPC to a subject. Exemplary “cholesterol storage disease ordisorders” include but are not limited to Niemann-Pick disease, type C1.Whether cholesterol storage and related pathophysiology may be impactedby NPC1 function in other conditions is certain and extends the utilityof NPC directed therapies, specifically NPC1 gene therapy, toward othermore common disorders in the future. For example, a subset ofneuropsychiatric disorders, such as dementia, seizures, andatherosclerotic brain disease might to be influenced by or improvedafter cholesterol reduction mediated by NPC1 activity and as such, thesegroups of patients might be candidates for NPC1 viral gene therapy.

Terms Relating to Molecular Biology

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual. Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach. Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization; B. D. Hames & S. J. Higgins eds.(1985); Transcription And Translation; [B. D. Hames & S. J. Higgins,eds. (1984); Animal Cell Culture; R. I. Freshney, ed. (1986);Immobilized Cells And Enzymes; IRL Press, (1986); B. Perbal, A PracticalGuide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

As used herein, the term “isolated” means that the referenced materialis removed from the environment in which it is normally found. Thus, anisolated biological material can be free of cellular components, i.e.,components of the cells in which the material is found or produced. Inthe case of nucleic acid molecules, an isolated nucleic acid includes aPCR product, an isolated mRNA, a cDNA, or a restriction fragment. Inanother embodiment, an isolated nucleic acid is preferably excised fromthe chromosome in which it may be found, and more preferably is nolonger joined to non-regulatory, non-coding regions, or to other genes,located upstream or downstream of the gene contained by the isolatednucleic acid molecule when found in the chromosome. In yet anotherembodiment, the isolated nucleic acid lacks one or more introns.Isolated nucleic acid molecules include sequences inserted intoplasmids, cosmids, artificial chromosomes, and the like. Thus, in aspecific embodiment, a recombinant nucleic acid is an isolated nucleicacid. An isolated protein, may be associated with other proteins ornucleic acids, or both, with which it associates in the cell, or withcellular membranes if it is a membrane-associated protein. In a specificembodiment, an isolated NPC1 protein is a recombinant NPC1 proteinexpressed from an expression vector. An isolated material may be, butneed not be, purified.

As used herein, the term “cDNA” (complementary DNA) refers to a piece ofDNA lacking internal, non-coding segments (introns) and regulatorysequences that determine transcription. cDNA can be synthesized in thelaboratory by reverse transcription from messenger RNA extracted fromcells.

As used herein, the term “ORF” (open reading frame) refers to a seriesof nucleotide triplets (codons) coding for amino acids without anytermination codons. These sequences are usually translatable into apeptide.

As used herein, the term “ortholog” refers to two nucleotide sequencesthat share a common ancestral sequence and diverged when a speciescarrying that ancestral sequence split into two species. Orthologoussequences are also homologous sequences.

As used herein, the terms “probes” and “primers” refers tooligonucleotide sequences that may readily be prepared based on thenucleic acids provided by this invention. A probe comprises an isolatednucleic acid attached to a detectable label or reporter molecule.Typical labels include radioactive isotopes, ligands, chemiluminescentagents, and enzymes. Methods for labeling and guidance in the choice oflabels appropriate for various purposes are discussed. e.g., in Sambrooket al. (1989) and Ausubel et al. (1987). “Primers” are short nucleicacids, preferably DNA oligonucleotides 15 nucleotides or more in length.Primers may be annealed to a complementary target DNA strand by nucleicacid hybridization to form a hybrid between the primer and the targetDNA strand, and then extended along the target DNA strand by a DNApolymerase enzyme. Primer pairs can be used for amplification of anucleic acid sequence, e.g., by the polymerase chain reaction (PCR) orother nucleic-acid amplification methods known in the art.

Methods for preparing and using probes and primers are described, forexample, in Sambrook et al. (1989), Ausubel et al. (1987), and Innis etal., (1990). PCR primer pairs can be derived from a known sequence, forexample, by using computer programs intended for that purpose such asPrimer (Version 0.5, 1991, Whitehead Institute for Biomedical Research,Cambridge, Mass.). One of skill in the art will appreciate that thespecificity of a particular probe or primer increases with its length.Thus, for example, a primer comprising 20 consecutive nucleotides of thehuman NPC1 cDNA or gene will anneal to a-target sequence such as an NPC1gene homolog from rat contained within a genomic rat genomic DNA librarywith a higher specificity than a corresponding primer of only 15nucleotides. Thus, in order to obtain greater specificity, probes andprimers may be selected that comprise 20, 25, 30, 35, 40, 50 or moreconsecutive nucleotides of the NPC1 cDNA or gene sequences.

The invention thus includes isolated nucleic acid molecules thatcomprise specified lengths of the disclosed NPC1 DNA (or cDNA) or genesequences. Such molecules may comprise at least 20, 25, 30, 35, 40 or 50consecutive nucleotides of these sequences and may be obtained from anyregion of the disclosed sequences.

As used herein, a “vector” nucleic acid molecule as introduced into ahost cell, thereby producing a transformed host cell. A vector mayinclude nucleic acid sequences that permit it to replicate in the hostcell, such as an origin of replication. A vector may also include one ormore selectable marker genes and other genetic elements known in theart. A vector may include a “gene transfer vector,” “gene therapyvector,” or “gene therapy construct,” or similar terms, which refer tospecific vector constructs that are suitable to conduct gene transfer toadminister a desired gene.

The terms “vector,” “cloning vector,” and “expression vector” mean thevehicle by which an ASM DNA or RNA sequence (e.g., a foreign gene) canbe introduced into a host cell so as to transform the host and promoteexpression (e.g., transcription and translation) of the introducedsequence. Vectors include any genetic element, such as a plasmid, phage,transposon, cosmid, chromosome, virus, virion, etc., which is capable ofreplication when associated with the proper control elements and whichcan transfer ASM gene sequences between cells. Thus, the term includescloning and expression vehicles, as well as viral vectors.

As used herein, the term “expression system” means a host cell andcompatible vector under suitable conditions, e.g., for the expression ofan NPC1 protein coded for by foreign DNA carried by the vector andintroduced to the host cell. Common expression systems include E. colihost cells and plasmid vectors, insect host cells such as Sf9, Hi5 or S2cells and Baculovirus vectors, and expression systems, and mammalianhost cells and vectors. The term “expression system” also may refer to asuitable gene therapy vector, which may be delivered by any means,including ex vivo and in vivo methods.

The term “host cell” means any cell of any organism that is selected,modified, transformed, grown, or used or manipulated in any way, for theproduction of a substance by the cell, for example the expression by thecell of a human ASM gene, including a DNA or RNA sequence, or the NPC1enzyme. Host cells can further be used for preliminary evaluation ofother assays. A “recombinant DNA molecule” is a DNA molecule that hasundergone a molecular biological manipulation or engineering. In oneembodiment of the invention, the host cell is a fibroblast.

A “gene” is a sequence of nucleotides that code for a “gene product”.Generally, a gene product is a protein. However, a gene product can alsobe another type of molecule in a cell, such as an RNA (e.g., a tRNA or arRNA). For the purposes of the present invention, a gene product alsorefers to an mRNA sequence which may be found in a cell. As used herein,a gene can refer to the nucleotide sequences encoding wild-type ormutant NPC1 or NPC2 genes.

As used herein, a “transformed cell” is a cell into which has beenintroduced a nucleic acid molecule by molecular biology techniques orgene therapy techniques. As used herein, the term transformationencompasses all techniques by which a nucleic acid molecule might beintroduced into such a cell, including transfection with viral vectors,transformation with plasmid vectors, and introduction of naked DNA byelectroporation, lipofection, and particle gun acceleration and includesboth in vitro and in vivo conditions.

As used herein, the term “purified” does not require absolute purity;rather, it is intended as a relative term. Thus, for example, a purifiedNPC1 protein preparation is one in which the NPC1 protein is more purethan the protein in its natural environment within a cell. Preferably, apreparation of an NPC1 protein is purified such that the NPC1 proteinrepresents at least 50% of the total protein content of the preparation.

As used herein, the term “operably linked” refers to where a firstnucleic acid sequence (e.g., an NPC1 gene) is operably linked with asecond nucleic acid sequence (e.g., a promoter sequence) when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

As used herein, the term “recombinant nucleic acid” is one that has asequence that is not naturally occurring or has a sequence that is madeby an artificial combination of two otherwise separated segments ofsequence. This artificial combination is often accomplished by chemicalsynthesis or, more commonly, by the artificial manipulation of isolatedsegments of nucleic acids, e.g., by genetic engineering techniques.

As used herein, the term “sequence identity” refers to the similaritybetween two nucleic acid sequences, or two amino acid sequences and isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs of the human and mouse NPC1 proteins will possess a relativelyhigh degree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well-known in theart. Various programs and alignment algorithms are described in: Smithand Waterman (1981); Needleman and Wunsch (1970); Pearson and Lipman(1988); Higgins and Sharp (1988); Higgins and Sharp (1989); Corpet etal. (1988); Huang et al. (1992); and Pearson et al. (1994). Altschul etal. (1994) presents a detailed consideration of sequence alignmentmethods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al.,1990) is available from several sources, including the National Centerfor Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet,for use in connection with the sequence analysis programs blastp,blastn, blastx, tblastn and tblastx. It can be accessed at the NCBIonline site under the “BLAST” heading. A description of how to determinesequence identity using this program is available at the NCBI onlinesite under the “BLAST overview” subheading.

Homologs of the disclosed NPC1 and NPC2 proteins are typicallycharacterized by possession of at least 70% sequence identity countedover the full length alignment with the disclosed amino acid sequence ofeither the human or mouse NPC1/NPC2 sequences using the NCBI Blast 2.0,gapped blastp set to default parameters. Proteins with even greatersimilarity to the reference sequences will show increasing percentageidentities when assessed by this method, such as at least 75%, at least80%, at least 90% or at least 95% sequence identity. When less than theentire sequence is being compared for sequence identity, homologs willtypically possess at least 75% sequence identity over short windows of10-20 amino acids, and may possess sequence identities of at least 85%or at least 90% or 95% depending on their similarity to the referencesequence. Methods for determining sequence identity over such shortwindows are described at the NCBI online site under the “FrequentlyAsked Questions” subheading. One of skill in the art will appreciatethat these sequence identity ranges are provided for guidance only; itis entirely possible that strongly significant homologs could beobtained that fall outside of the ranges provided. The present inventionprovides not only the peptide homologs are described above, but alsonucleic acid molecules that encode such homologs, such as thosegenerated by codon optimization. In an embodiment, changing thenucleotide sequence on the corresponding codons will generate asynthetic NPC1 or NPC2 gene that would have improved translationefficiency and detection in the presence of the endogenous gene.

One indication that two nucleic acid sequences are substantiallyidentical is that the polypeptide which the first nucleic acid encodesis immunologically cross reactive with the polypeptide encoded by thesecond nucleic acid (e.g., a human NPC1 protein and an NPC1 homolog fromanother species, or a variant human NPC1 protein).

Another indication that two nucleic acid sequences are substantiallyidentical is that the two molecules hybridize to each other understringent conditions. Stringent conditions are sequence dependent andare different under different environmental parameters. Generally,stringent conditions are selected to be about 5° C., to 20° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of the target sequencehybridizes to a perfectly matched probe. Conditions for nucleic acidhybridization and calculation of stringencies can be found in Sambrooket al. (1989) and Tijssen (1993) and are otherwise known in the art.

Terms Relating to Gene Therapy

The term “gene therapy” refers to a method of changing the expression ofan endogenous gene by exogenous administration of a gene, i.e., awildtype or mutant NPC1 or NPC2 gene. As used herein, gene therapy alsorefers to the replacement of a defective NPC1 or NPC2 gene, orreplacement of a missing NPC1 or NPC2 gene, by introducing a functionalgene or portion of a gene corresponding to the defective or missing NPC1or NPC2 gene into somatic or stem cells of an individual in need. Genetherapy can be accomplished by “ex vivo” methods, in whichdifferentiated or somatic stem cells are removed from the individual'sbody followed by the introduction of a normal copy of the defective geneinto the explanted cells using a viral vector as the gene deliveryvehicle. In addition, in vivo transfer involves direct gene transferinto cells in the individual in situ using a broad range of viralvectors (e.g., AAV), liposomes, nanoparticles, protein:DNA complexes,modified nucleic acids or naked DNA in order to achieve a therapeuticoutcome.

The term “transgene” refers to a polynucleotide that is introduced intoa cell of and is capable of being expressed under appropriate conditionsand confers a desired property to a cell into which it was introduced,or otherwise leads to a desired therapeutic outcome.

The terms “genome particles (gp),” or “genome equivalents,” or genomecopies (gc) as used in reference to a viral titer, refer to the numberof virions containing the recombinant AAV DNA genome, regardless ofinfectivity or functionality. The number of genome particles in aparticular vector preparation can be measured by procedures such asdescribed elsewhere herein, or for example, in Clark et al. (1999) Hum.Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272-278.

The terms “infection unit (iu),” “infectious particle,” or “replicationunit,” as used in reference to a viral titer, refer to the number ofinfectious and replication-competent recombinant AAV vector particles asmeasured by the infectious center assay, also known as replicationcenter assay, as described, for example, in McLaughlin et al. (1988) J.Virol., 62:1963-1973.

The term “transducing unit (tu)” as used in reference to a viral titer,refers to the number of infectious recombinant AAV vector particles thatresult in the production of a functional transgene product as measuredin functional assays such as described elsewhere herein, or for example,in Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or in Fisher et al.(1996) J. Virol., 70:520-532 (LFU assay).

Terms Relating to Therapeutic Application

The present invention further provides a method for the prevention ortreatment of Type A and Type B NPD, which method comprises increasingthe expression or activity of the mutant ASM enzyme, or by increasingthe activity of recombinant, wild-type replacement ASM enzyme, in asubject or patient in need of such treatment.

As used herein, the term “administering” is meant to refer to a means ofproviding the composition (e.g., to the subject in a manner that resultsin the composition being inside the subject's body. Such anadministration can be by any route including, without limitation,subcutaneous, intradermal, intravenous, intra-arterial, intraperitoneal,sublingual, buccal, and intramuscular. In certain embodiments, thedelivery may be appropriate for CNS delivery, e.g., epidural,intracerebral, or intracerebroventricular.

The invention provides a number of compositions (e.g., sequences andvectors) that are useful for the development of highly specific drugs totreat or prevent a disease or disorder in a subject, as furthercharacterized by the methods delineated herein. In addition, the methodsof the invention provide a facile means to identify therapies that aresafe for use in subjects. Other disorders that can feature cholesterolstorage are contemplated, including adult forms of dementia andconditions that may be caused, in part, by diminished activity of NPC1.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agents.The compositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see Martin Remington'sPharm. Sci., 15th Ed. (Mack Publ. Co., Easton (1975)).

A “subject” or “patient” is a human or an animal that has developed, oris likely to develop NPC disease, more particularly a mammal, preferablya rodent or a primate, and most preferably a human. In one embodiment,the patient is a member of the Ashkenazi Jewish population who has beendiagnosed with, or who has been identified as having an increased riskof developing NPC disease due to inherited mutations in the NPC1 or NPC2gene. In another embodiment, the patient is a member of the FrenchCanadian population of Nova Scotia, an inhabitant of the Maghreb region(Tunisia, Morocco, Algeria) of North Africa, or a member of theSpanish-American population of southern New Mexico and Colorado.However, Niemann-Pick disease is pan-ethnic, and the term subjectencompasses anyone in the world having, or genetically at risk ofdeveloping, NPC disease. The term “in vitro” has its art recognizedmeaning, e.g., involving purified reagents or extracts, e.g., cellextracts. The term “in vivo” also has its art recognized meaning, e.g.,involving living cells, e.g., immortalized cells, primary cells, celllines, and/or cells in an organism.

“Treatment”, or “treating” as used herein, is defined as the applicationor administration of a therapeutic agent (e.g., an AAV-NPC vector) to apatient, or application or administration of a therapeutic agent to anisolated tissue or cell line from a patient, who has a disorder with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease or disorder, or symptoms of the disease ordisorder. The term “treatment” or “treating” is also used herein in thecontext of administering agents prophylactically. In the context of thepresent invention, the symptoms that may be alleviated can include, butare not limited to, the accumulation of sphingomyelin inreticuloendothelial lysosomes, which results in hepatosplenomegaly,psychomotor retardation, pulmonary abnormalities, progressiveneurodegeneration. In some instances, treatment will prevent deathresulting from NPC disease.

The term “prevention” refers to the prevention of the onset of thedisease, which means to prophylactically interfere with a pathologicalmechanism that results in the disease. In the context of the presentinvention, such a pathological mechanism can be an increase expressionof mutant NPC1 or NPC2.

The terms “effective dose” or “effective dosage” or “therapeuticallyeffective amount” are defined as an amount sufficient to achieve or atleast partially achieve the desired effect. The term “therapeuticallyeffective dose” is defined as an amount sufficient to cure or at leastpartially arrest the disease and its complications in a patient alreadysuffering from the disease or prevent the disease prophylactically. Theterm “patient” includes human and other mammalian subjects that receiveeither prophylactic or therapeutic treatment. As it pertains to theinstant invention, the term “therapeutically effective amount” also isused herein to mean an amount or dose of a gene therapy vector encodingNPC1 or NPC2 (or a mutant or functional variant thereof) sufficient toincrease the level of NPC1 or NPC2 activity over the mutant or defectivelevel to about 3-5%, preferably by about 10%, and more preferably byabout 30%, or about 40%, or about 50%, or about 60%, or about 70%, orabout 80%, or about 90%, or about 95%, or even up to 100% of the levelfound in normal cells. Preferably, a therapeutically effective amountcan ameliorate or prevent a clinically significant deficit in NPC1 orNPC2 in the subject. Alternatively, a therapeutically effective amountis sufficient to cause an improvement in a clinically significantcondition in the subject, e.g., amelioration of progressiveneurodegeneration in Type C NPD patients.

Certain methodologies of the instant invention include at least one stepthat involves comparing a value, level, feature, characteristic,property, etc, to a “suitable control”, referred to interchangeablyherein as an “appropriate control”. A “suitable control” or “appropriatecontrol” is a control or standard familiar to one of ordinary skill inthe art useful for comparison purposes. In one embodiment, a “suitablecontrol” or “appropriate control” is a value, level, feature,characteristic, property, etc. determined prior to performing a genetherapy methodology, as described herein. For example, a transcriptionrate, mRNA level, translation rate, protein level, biological activity,cellular characteristic or property, genotype, phenotype, etc. can bedetermined prior to introducing an AAV or other vector of the inventioninto a cell or organism. In another embodiment, a “suitable control” or“appropriate control” is a value, level, feature, characteristic,property, etc. determined in a cell or organism, e.g., a control ornormal cell or organism, exhibiting, for example, normal traits. In yetanother embodiment, a “suitable control” or “appropriate control” is apredefined value, level, feature, characteristic, property, etc.

Other definitions appear in context throughout the disclosure.

Gene Therapy Vectors

In one aspect, the present invention relates to gene therapy vectors orconstructs comprising NPC1 and/or NPC2 genes, or derivatives and/ormutants thereof, which are operably linked to at least a promoterelement that is capable of being expressed in a tissue of the centralnervous system. In certain embodiments, the promoter is aneuronal-specific calmodulin promoter or derivative thereof. In otherembodiments, the promoter is a constitutive promoter, e.g., an EF1αconstitutive promoter or derivative thereof, which is capable of beingexpressed in neuronal tissues. As demonstrated by a reduction topractice using accepted NPC mouse models, the gene therapy vectors ofthe present invention were effective in treating and/or preventing NPC.

In certain embodiments, the gene therapy vectors or constructs comprisean NPC1 gene, or a derivative and/or mutant NPC1 gene. The NPC1 gene,including any derivatives and/or mutants thereof, can encode a wildtypeNPC1 polypeptide, any a functional fragment or variant thereof. Thevariants or functional fragments of NPC1 may have increased or decreasedactivity as compared to a wildtype NPC1 protein, or the activity may beunchanged.

In certain other embodiments, the gene therapy vectors or constructscomprise an NPC2 gene, or a derivative and/or mutant NPC2 gene. The NPC2gene, including any derivatives and/or mutants thereof, can encode awildtype NPC2 polypeptide, any a functional fragment or variant thereof.The variants or functional fragments of NPC2 may have increased ordecreased activity as compared to a wildtype NPC2 protein, or theactivity may be unchanged.

The NPC1 and/or NPC2 genes or nucleotide sequences comprising a codingregion for NPC1 and/or NPC2 proteins may be obtained from any source,including human, mouse, horse, pig, monkey, and the like. The nucleotidesequences encoding NPC1 and/or NPC2 in human, and NPC1 and/or NPC2homologs from species other than human, are generally known in the artand can be obtained from public sequence repositories, including, forexample, GenBank. In particular, cDNA sequences encoding NPC1 and/orNPC2 proteins (or variants thereof) may also be obtained from publicsequence repositories such as GenBank.

For example, the following NPC1 sequences (or any variants comprising orgenetically modified to comprise any mutations that encode a functionalvariant NPC1) are contemplated for use in the gene therapy constructs ofthe present invention:

GenBank Accession No. BC063302 (Homo sapiens Niemann-Pick disease, typeC1, mRNA (cDNA clone), which provides the NPC1 cDNA coding sequence (SEQID NO: 10, FIG. 13) and the NPC1 polypeptide amino acid sequence (SEQ IDNO: 9, FIG. 12);

GenBank Accession No. BC117178 (Homo sapiens NPC1 (Niemann-Pick disease,type C1, gene)-like 1 mRNA (cDNA clone), which provides a variant NPC1cDNA coding sequence and NPC1 polypeptide amino sequence;

GenBank Accession No. BC143756 (Homo sapiens NPC1 (Niemann-Pick disease,type C1, gene)-like 1, mRNA (cDNA clone), which provides a variant NPC1cDNA coding sequence and NPC1 polypeptide amino sequence;

GenBank Accession No. AF258783.1 (Felis catus Niemann-Pick type C1disease protein (NPC1) mRNA, complete cds) which provides cat NPC1 cDNAcoding sequence and cat NPC1 polypeptide amino sequence;

GenBank Accession No. BC054539 (Mouse Npc1 (Niemann-Pick disease, typeC1, gene) mRNA (cDNA clone), which provides mouse Npc1 cDNA codingsequence and mouse NPC1 polypeptide amino sequence;

GenBank Accession No. BC151276 (Bovine NPC1 (Niemann-Pick disease, typeC1, gene) mRNA (cDNA clone), which provides bovine NPC1 cDNA codingsequence and bovine NPC1 polypeptide amino sequence; and

GenBank Accession No. BC090541 (Zebrafish NPC1 (Niemann-Pick disease,type C1, gene) mRNA (cDNA clone), which provides Zebrafish NPC1 cDNAcoding sequence and Zebrafish NPC1 polypeptide amino sequence.

The disclosed subject matter further encompasses any NPC1 gene and/orpolypeptide sequence not expressly indicated here, but which is publiclyavailable at the time of the present invention, or which becomesavailable after the time of the invention.

For example, the following NPC2 sequences (or any variants comprising orgenetically modified to comprise any mutations that encode a functionalvariant NPC2) are contemplated for use in the gene therapy constructs ofthe present invention:

GenBank Accession No. BC002532 (Homo sapiens Niemann-Pick disease, typeC2, mRNA (cDNA clone), which provides the NPC2 cDNA coding sequence (SEQID NO: 12, FIG. 15) and the NPC2 polypeptide amino acid sequence (SEQ IDNO: 11, FIG. 14);

GenBank Accession No. KJ893081 (Synthetic construct Homo sapiens cloneccsbBroadEn_02475 NPC2 gene, encodes complete protein), which providesthe NPC2 cDNA coding sequence and the NPC2 polypeptide amino acidsequence;

GenBank Accession No. BC045895 (Zebrafish Niemann-Pick disease, type C2,mRNA (cDNA clone), which provides the Zebrafish NPC2 cDNA codingsequence and the Zebrafish NPC2 polypeptide amino acid sequence;

GenBank Accession No. NM_173918 (Bovine Niemann-Pick disease, type C2,mRNA (cDNA clone), which provides the Bovine NPC2 cDNA coding sequenceand the Bovine NPC2 polypeptide amino acid sequence;

GenBank Accession No. BC102504 (Bovine Niemann-Pick disease, type C2,mRNA (cDNA clone), which provides the Bovine NPC2 cDNA coding sequenceand the Bovine NPC2 polypeptide amino acid sequence; and

GenBank Accession No. NM_214206 (Pig Niemann-Pick disease, type C2, mRNA(cDNA clone), which provides the pig NPC2 cDNA coding sequence and thePig NPC2 polypeptide amino acid sequence.

The disclosed subject matter further encompasses any NPC2 gene and/orpolypeptide sequence not expressly indicated here, but which is publiclyavailable at the time of the present invention, or which becomesavailable after the time of the invention.

In preferred embodiments, the transgene encodes a biologically activemolecule, expression of which in the subject, e.g., in the CNS of asubject, results in at least partial correction of the cholesterolstorage disease or disorder, for example. Niemann-Pick disease, Type C.In some embodiments, the transgene encodes NPC1 (or a functional variantand/or fragment thereof). In other embodiments, the transgene encodesNPC2 (or a functional variant and/or fragment thereof). The genomic andfunctional mRNA, cDNA and corresponding polypeptide sequences of human,mouse, or other species NPC1 and NPC2 genes and proteins are known, asindicated above, and in particular are available as GenBank AccessionNos.: NM_000271.4; NM_008720.2; NM_006432.3; NM_023409.4; andcorresponding polypeptides NP_000262.2; NP_032746.2; NP_006423.1; andNP_075898.1.

Nucleotide sequences encoding NPC1 or NPC2 genes (or variants thereof)may be obtained by any known molecular biology technique, including bycloning, synthesis, or PCR amplification. Oligonucleotides for using inamplification reactions and/or probes for use in gene cloning may besynthesized or otherwise obtained by any known means and based on thenucleotide sequences that flank the desired gene or coding regionencoding the NPC1 or NPC2 target genes. Methods and techniques for genecloning and/or PCR amplification are well known in the art and arediscussed elsewhere herein.

The gene therapy constructs described herein also comprise a vector (orgene therapy expression vector) into which the gene of interest (e.g.,NPC1 or NPC2 gene) is cloned or otherwise which includes the gene ofinterest in a manner such that the nucleotide sequences of the vectorallow for the expression (constitutive or otherwise regulated in somemanner) of the gene of interest. The vector constructs herein describedinclude any suitable gene expression vector that is capable of beingdelivered to a tissue of interest (e.g., CNS) and which will provide forthe expression of the gene of interest in the selected tissue ofinterest (e.g., CNS). In a preferred embodiment, the gene therapy vectoris capable of efficient delivery to a tissue of the central nervoussystem, including the spine and the brain, and in particular, is capableof crossing the blood-brain barrier of the brain.

In a preferred embodiment, the vector is an adeno-associated virus (AAV)vector because of the capacity of AAV vectors to cross the blood-brainbarrier and transduction of neuronal tissue. In the methods disclosedherein, AAV of any serotype can be used, though in certain embodiments,it is advantageous to use a vector that is capable of undergoingretrograde axonal transport in a disease-compromised brain. The serotypeof the viral vector used in certain embodiments of the invention isselected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh33, AAV rh34, AAV Anc80,AAV PHP.B, and others (see, e.g., Cao et al. (2002) PNAS,99:11854-11859; and Viral Vectors for Gene Therapy: Methods andProtocols, ed. Machida, Humana Press, 2003, incorporated herein byreference). Other serotype besides those listed herein are alsocontemplated. In certain exemplary embodiments, AAV 2/9 is used. Theherein disclosed compositions and methods may also use AAV chimericvectors, whereby portions of AAV are fused with other similar vectors,such as Adenovirus.

AAV vectors are derived from single-stranded (ss) DNA parvoviruses thatare nonpathogenic for mammals (reviewed in Muzyscka (1992) Curr. Top.Microb. Immunol., 158:97-129, incorporated herein by reference).Briefly. AAV-based vectors have the rep and cap viral genes that accountfor 96% of the viral genome removed, leaving the two flanking145-basepair (bp) inverted terminal repeats (ITRs), which are used toinitiate viral DNA replication, packaging and integration. In theabsence of helper virus, wild-type AAV integrates into the humanhost-cell genome with preferential site-specificity at chromosome 19q13.3 or it may remain expressed episomally. A single AAV particle canaccommodate up to 5 kb of ssDNA, therefore leaving about 4.5 kb for atransgene and regulatory elements, which is typically sufficient.However, trans-splicing systems as described, for example, in U.S. Pat.No. 6,544,785, may nearly double this limit.

In an illustrative embodiment, the AAV backbone, comprising sequencesbetween two AAV inverted terminal repeats (ITRs), is pseudotyped usingthe serotype 2 capsid to create an AAV2 vector. Adeno-associated virusof many serotypes, especially AAV2, have been extensively studied andcharacterized as gene therapy vectors. Those skilled in the art will befamiliar with the preparation of functional AAV-based gene therapyvectors. Numerous references to various methods of AAV production,purification and preparation for administration to human subjects can befound in the extensive body of published literature (see, e.g., ViralVectors for Gene Therapy: Methods and Protocols, ed. Machida, HumanaPress, 2003, incorporated herein by reference). Additionally, AAV-basedgene therapy targeted to cells of the CNS has been described in U.S.Pat. Nos. 6,180,613 and 6,503,888 (each of which are incorporated hereinby reference).

Optionally, the AAV viral capsid is AAV2/9, AAV9, AAVrh8, AAVrh10, AAVAnc80, or AAV PHP.B; however, the serotype of the viral capsid used incertain embodiments of the invention can be selected from among knownviral capsids, including AAV viral capsids of other known serotypes.

Optionally, the gene therapy vector, e.g., AAV or AAV-based vector, canbe modified to improve virus uptake into the target tissue of interest(e.g., CNS), viral stability, and tropism. For example, the capsid of anAAV vector may be modified with a ligand (e.g., synthetic or naturallyoccurring small molecule, peptide, or polypeptide, or other biomolecule)that binds to a receptor at or in the tissue of interest (e.g., CNS).Other modification are possible to improve and/or enhance the functionalproperties of the vector being used to both target the tissue ofinterest and allow the construct to enter and effectively transduce thetarget cells. Such modifications will be within the skill set of aperson having ordinary skill in the art.

Further information regarding the use of AAV vectors can be found in theart, for example, in Kaplitt et al. (1994) Nat. Genet., 8:148-154;Bartlett et al. (1998) Hum. Gene Ther., 9:1181-1186; and Passini et al.(2002) J. Neurosci., 22:6437-6446, each of which are incorporated hereinby reference. Furthermore, these viral vectors can transduce a varietyof CNS cell types, including neurons, when delivered by the systemicroute, intrathecal route or by direct brain injection.

As further contemplated herein, the gene therapy vectors may comprise atransgene (e.g., NPC1 or NPC2) that is operably linked to a promoter orother genetic transcriptional and/or translational control elements.Certain AAV vectors pre-engineered with or comprising promoters can beobtained from public sources, including, for examplewww.vectorbiolabs.com or www.addgene.org, which are incorporated hereinby reference.

In certain embodiments, the promoter is promoter which is capable ofefficient inducible expression in the CNS. In still other embodiments,the promoter is constitutively active in the CNS. In certain preferredembodiments, the promoter provides for selective expression in the CNS,and expression outside of the CNS is limited or entirely absent.Promoter sequences having differing characteristics and expressionprofiles are well known in the art, including those that aretissue-specific, tissue-non-specific, constitutive, and inducible.Reference can be further made to, for example, Papadakis et al.,“Promoters and Control Elements: Designing Expression Cassettes for GeneTherapy.” Current Gene Therapy, 2004, 4, 89-113, the contents of whichare incorporated herein by reference. Promoters contemplated by thepresent invention include, but are not limited to: Apo A-I, ApoE,serpina (TBG), alpha-1-antitrypsin (hAAT) (liver specific); MCK (musclespecific); GFAP, NSE, Synapsin I, Preproenkephalin, Dopamineb-hydroxylase (dbH), Prolactin, Myelin basic protein(neuronal-specific), GUSB, CBA, CAG and Ankyrin (erythroid specific).

In a particular embodiment, the disclosed compositions and methodsutilize an AAV vector in conjunction with a _(mini)-calmodulin promoter(aka CaMKII or _(mini)CaMKII promoter) (SEQ ID NO: 3. FIG. 4) whereCaMKII or _(mini)CaMKII can be from human or mouse or any other mammaland has neuronal-specific tropism and expression characteristics.

In another particular embodiment, the disclosed compositions and methodsutilize an AAV vector in conjunction with an elongation factor 1-alphapromoter (aka EF1α promoter) (SEQ ID NO: 8, FIG. 11) which isconstitutive and expresses in neuronal tissues. For the purpose of thisdocument EF1α can be intact or truncated and terms EF1α or EF1α (short)or _(mini)EF1α can be used interchangeably. Also, EF1α is same as Ef1αand can be obtained from human or mouse.

Methods of Treatment

In one aspect, the present invention provides methods for treating acholesterol storage disease or disorder in mammals, such as Neimann-Pickdisease, Type C. In preferred embodiments, the populations treated bythe methods of the invention include, but are not limited to, patientshaving or at risk for developing a cholesterol storage disease ordisorder, e.g., Niemann-Pick disease, type C1, particularly, if such adisease affects the CNS. In an illustrative embodiment, the disease isNiemann-Pick disease, Type C1.

In certain aspects of the invention, the method of treating acholesterol storage disease or disorder comprises administration of ahigh titer gene therapy vector described herein (e.g., an AAV-based genetherapy vector) carrying a therapeutic transgene so that the transgeneproduct is expressed at a therapeutic level in the CNS of a subject. Insome embodiments, the viral titer of the composition is at least: (a) 5,6, 7, 8, 8.4, 9, 9.3, 10, 15, 20, 25, or 50×10¹² gc/ml; (b) 5, 6, 7, 8,8.4, 9, 9.3, 10, 15, 20, 25, or 50×10⁹ tu/ml; or (c) 5, 6, 7, 8, 8.4, 9,9.3, 10, 15, 20, 25, or 50×10¹⁰ iu/ml. In further embodiments, theadministration is accomplished by direct intraparenchymal injection ofsolution comprising a high titer gene therapy vector described herein(e.g., an AAV-based gene therapy vector) into the diseased brain,thereafter the transgene is expressed distally, contralaterally oripsilaterally, to the administration site at a therapeutic level atleast 2, 3, 5, 8 10, 15, 20, 25, 30, 35, 40, 45, or 50 mm from theadministration site.

In further embodiments, the administration is accomplished by directintrathecal injection of a solution comprising a high titer gene therapyvector described herein (e.g., an AAV-based gene therapy vector) intothe spinal fluid compartment, as is routine for practioners of the art,and thereafter the transgene is expressed distally, contralaterally,ipsilaterally and globally in the CNS, to the administration site at atherapeutic level at least 2, 3, 5, 8 10, 15, 20, 25, 30, 35, 40, 45, or50 mm from the administration site.

In certain embodiments, the transgene product (e.g., NPC1 or NPC2polypeptide) is expressed at a therapeutic level in a second site withinthe CNS distal to the first site. The distance between the first and thesecond sites is defined as the minimal distance region between the siteof administration (first site) and the boundary of the detectabletransduction of the distal site (second site) as measured usingprocedures known in the art, e.g., magnetic resonance imaging includingspectroscopy or direct brain biopsy. Some neurons in the CNS of largermammals may span large distances by virtue of their axonal projections.For example, in humans, some axons may span a distance of 1000 mm orgreater. Thus, in various methods of the invention, a gene therapyvector of the invention can be axonally transported along the entirelength of the axon at such a distance to reach and transduce the parentcell body.

A site of vector administration within the CNS can be chosen based onthe desired target region of neuropathology and, optionally, thetopology of brain circuits involved when an administration site and thetarget region have axonal connections. In certain embodiments, thetarget region can be defined, for example, using 3-D stereotaxiccoordinates. In some embodiments, the administration site is chosen sothat at least 0.1, 0.5, 1, 5, or 10% of the total amount of vectorinjected is delivered distally at the target region of at least 1, 200,500, or 1000 mm³. An administration site may be localized in a regioninnervated by projection neurons connecting distal regions of the brain.For example, the substantia nigra and ventral tegmental area send denseprojections to the caudate and putamen (collectively known as thestriatum). Neurons within the substantia nigra and ventral tegmentum canbe targeted for transduction by retrograde transport of a gene therapyconstruct described herein (e.g., AAV based vector) following injectioninto the striatum. As another example, the hippocampus receiveswell-defined, predictable axonal projections from other regions of thebrain. Other administration sites may be localized, for example, in thespinal cord, brainstem (medulla and pons), mesencephalon, cerebellum,diencephalon (thalamus, hypothalamus), telencephalon (corpus striatum,cerebral cortex, or, within the cortex, the occipital, temporal,parietal or frontal lobes), or combinations thereof.

For identification of structures in the human brain, see, e.g., TheHuman Brain: Surface, Three-Dimensional Sectional Anatomy With MRI, andBlood Supply 2nd ed., eds. Deuteron et al., Springer Vela, 1999; Atlasof the Human Brain, eds. Mai et al., Academic Press; 1997; and Co-PlanarSterotaxic Atlas of the Human Brain: 3-Dimensional Proportional System:An Approach to Cerebral Imaging, eds. Tamarack et al., Thyme MedicalPub., 1988. For identification of structures in the mouse brain, see,e.g., The Mouse Brain in Sterotaxic Coordinates, 2nd ed., AcademicPress, 2000. If desired, the human brain structure can be correlated tosimilar structures in the brain of another mammal. For example, mostmammals, including humans and rodents, show a similar topographicalorganization of the entorhinal-hippocampus projections, with neurons inthe lateral part of both the lateral and medial entorhinal cortexprojecting to the dorsal part or septal pole of the hippocampus, whereasthe projection to the ventral hippocampus originates primarily fromneurons in medial parts of the entorhinal cortex (Principles of NeuralScience, 4th ed., eds Kandel et al., McGraw-Hill, 1991; The Rat NervousSystem, 2nd ed., ed. Paxinos, Academic Press, 1995). Furthermore, layerII cells of the entorhinal cortex project to the dentate gyrus, and theyterminate in the outer two-thirds of the molecular layer of the dentategyrus. The axons from layer III cells project bilaterally to the cornuammonis areas CA1 and CA3 of the hippocampus, terminating in the stratumlacunose molecular layer.

In certain embodiments, the target site can be located any region of theCNS, including the brain and the spinal cord, that contains a neuronsthat project to the first (administration) site. In some embodiments,the second site is in a region of the CNS chosen from the substantianigra, the medulla oblongata, or the spinal cord.

To deliver a gene therapy vector described herein specifically to aparticular region of the central nervous system, especially to aparticular region of the brain, it may be administered by sterotaxicmicroinjection. For example, on the day of surgery, patients will havethe sterotaxic frame base fixed in place (screwed into the skull). Thebrain with sterotaxic frame base (MRI-compatible with fiduciarymarkings) will be imaged using high resolution MRI. The MRI images willthen be transferred to a computer that runs stereotaxic software. Aseries of coronal, sagittal and axial images will be used to determinethe target site of vector injection, and trajectory. The softwaredirectly translates the trajectory into 3-dimensional coordinatesappropriate for the stereotaxic frame. Burr holes are drilled above theentry site and the stereotaxic apparatus localized with the needleimplanted at the given depth. The vector in a pharmaceuticallyacceptable carrier will then be injected. The AAV vector is thenadministrated by direct injection to the primary target site andretrogradely transported to distal target sites via axons. Additionalroutes of administration may be used, e.g., superficial corticalapplication under direct visualization, or other non-stereotaxicapplication.

Optionally, non-CNS delivery can also be performed, e.g., forcholesterol storage diseases or disorders where non-CNS delivery wouldalso be desirable. Such non-CNS delivery of the compositions (e.g.,constructs) of the instant invention can be performed in addition to oras an alternative to CNS delivery. In certain such embodiments,injection, e.g., intravenous, intraperitoneal, etc. injection can beperformed using the compositions of the instant invention. Directdelivery to large peripheral nerves is also considered.

In yet another method, a suitable AAV vector configured to express NPC1or NPC2 can be encapsidated with a capsid known to afford transductionof the blood brain barrier and further penetration of the CNS and itselements. In this embodiment, the AAV vector can be deliveredsystemically, by IV infusion, and engender both peripheral and CNScorrection, depending upon the promoter and serotype of the vector.

The total volume of material to be administered, and the total number ofvector particles to be administered, will be determined by those skilledin the art based upon known aspects of gene therapy. Therapeuticeffectiveness and safety can be tested in an appropriate animal model.For example, for NPC, in any Npc1^(−/−) model mouse such as theNpc^(nih) homozygous mice.

In experimental mice, the total volume of injected vector, e.g., AAVvector, solution is, for example, between 1 to 10 μl. For other mammals,including the human brain, volumes and delivery rates are appropriatelyscaled. For example, it has been demonstrated that volumes of 150 μl canbe safely injected in the primate brain (Janson et al. (2002) Hum. GeneTher., 13:1391-1412). Treatment may consist of a single injection pertarget site, or may be repeated along the injection tract, if necessary.Multiple injection sites can be used. For example, in some embodiments,in addition to the first administration site, a composition comprising agene therapy vector described herein carrying a transgene isadministered to another site that can be contralateral or ipsilateral tothe first administration site.

In another aspect, the invention provides a method of delivering atransgene product to a target cell of the CNS, which is a neuron or aglial cell, in a mammal afflicted with a cholesterol storage disease ordisorder, e.g., Niemann-Pick disease, type C. The method comprisescontacting an axonal ending of a neuron with a composition comprising anAAV vector carrying at least a part of a gene encoding a therapeutictransgene product, e.g., NPC1; allowing the viral particles to beendocytosed and retrogradely transported intracellularly along the axonto the nucleus of the neuron; allowing the transgene product to beexpressed and transported within the membrane(s) of the neuron, whereinthe transgene product thereby alleviates pathology related tocholesterol storage. In some embodiments, the concentration of the AAVvector in the composition is at least: (a) 5, 6, 7, 8, 8.4, 9, 9.3, 10,15, 20, 25, or 50×10¹² gc/ml; (b) 5, 6, 7, 8, 8.4, 9, 9.3, 10, 15, 20,25, or 50×10⁹ tu/ml; or (c) 5, 6, 7, 8, 8.4, 9, 9.3, 10, 15, 20, 25, or50×10¹⁰ iu/ml.

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a diseaseor disorder caused, in whole or in part, by altered cholesterol storage,optionally treatable via selective or systemic delivery of a NPC1-and/or NPC2-containing gene therapy vector to a subject.

In certain aspects, the invention provides a method for preventing in asubject, a disease or disorder as described herein (including, e.g.,NPC), by administering to the subject a gene therapy composition.Subjects at risk for the disease can be identified by, for example, oneor a combination of diagnostic or prognostic assays known in the art(e.g., genetic assessment of the subject and/or phenotypic assessment).Administration of a prophylactic agent can occur prior to the detectionof, e.g., NPC in a subject, or the manifestation of symptomscharacteristic of the disease or disorder, such that the disease ordisorder is prevented or, alternatively, delayed in its progression.

Another aspect of the invention pertains to methods of treating subjectstherapeutically, i.e., altering the onset of symptoms of the disease ordisorder. These methods can be performed in vitro (e.g., by culturingthe cell with the gene therapy composition) or, alternatively, in vivo(e.g., by administering the gene therapy composition to a subject).

With regard to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with the gene therapy transgene ofthe present invention to that individual's drug response genotype.Pharmacogenomics allows a clinician or physician to target prophylacticor therapeutic treatments to patients who will most benefit from thetreatment and to avoid treatment of patients who will experience toxicdrug-related side effects.

Gene Therapy Compositions

The invention, in part, pertains to a gene therapy compositioncomprising the NPC-providing vectors as described herein. The genetherapy composition of the invention can gain entry into a cell ortissue, e.g., a CNS cell or tissue, for treating or preventing NPCdisease or mitigating the complications, such as liver disease,neurological decline or seizures.

Advantageously, the gene therapy composition of the invention providesfor a controlled delivery of an active gene, especially a therapeuticgene, to a site of action at an optimum rate and therapeutic dose. Thus,improvements in therapeutic index may be obtained by modulating thedistribution of the active ingredient in the body and/or by modulatingthe promoter used in such gene therapy construct. Association of thegene therapy vector and/or viral vector containing such gene therapyvector with a delivery system enables, in particular, its specificdelivery to the site of action or its controlled expression of a geneafter targeting the action site. By reducing the amount of active genetherapy vector distributes to any compartments in which its presence isnot desired, it is possible to increase the efficacy of the gene therapyagent, and to reduce any toxic side effects or even modify or restoreactivity of gene therapy agents. In this application, the capsidserotype can influence route of delivery, cellular transductionefficacy, and dose required for a therapeutic effect. The promoter ofthe vector further dictates cell type expression i.e., in all cells oronly neurons and the degree to which expression occurs at the cellularlevel. As such, some promoters are stronger than others, and producehigher transgene expression. In another embodiment, microRNA (miRNA)binding sites are embedded in the 3′ untranslated region of thetherapeutic transgene to provide a cell specific inhibition oftranslation if the NPC transgene product is toxic in one cell typecompared to another. This approach would minimize off target expressionin cell types other than neurons if needed.

The invention also relates to pharmaceutical or diagnostic compositionscomprising the NPC-including vectors of the invention and apharmaceutically acceptable carrier. As such, direct RNA or DNA ormodified forms if such, including peptide or covalently modified nucleicacids, injections in the brain or other locations are considered usingthe therapeutic transgenes described in this application. In anotherembodiment, nanoparticles containing nucleic acids encoding NPC1 areused for gene delivery. The phrase “pharmaceutically acceptable carrier”is art recognized and includes a pharmaceutically acceptable material,composition or vehicle, suitable for administering compounds used in themethods described herein to subjects, e.g., mammals. The carriersinclude liquid or solid filler, diluent, excipient, solvent orencapsulating material, involved in carrying or transporting the subjectagent from one organ, or portion of the body, to another organ, orportion of the body. Each carrier must be “acceptable” in the sense ofbeing compatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;phosphate buffer solutions; and other non-toxic compatible substancesemployed in pharmaceutical formulations. Suitable pharmaceuticalcarriers are described in Remington's Pharmaceutical Sciences, MackPublishing Company, a standard reference text in this field.

In certain embodiments, the present invention provides for a viralvector composition comprising a gene therapy agent (e.g., NPC1 or NPC2operably linked to a tissue-specific or systemic promoter, optionallywithin a plasmid corresponding to the form of viral delivery systememployed, e.g., AAV viral vector plasmid) of the present invention. Theactive viral vector can be suitably formulated and introduced into theenvironment of the cell by any means that allows for a sufficientportion of the sample to enter the cell to induce expression of the genetherapy agent, if it is to occur. Many formulations for AAV and othervector-based gene therapy delivery are known in the art and can be used.

Such compositions can include the gene therapy agent and apharmaceutically acceptable carrier. Supplementary active compounds canalso be incorporated into the compositions. The AAV capsid can likewisebe modified to improve uptake and viral stability, and alter trophism.

A gene therapy composition can be formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intracranial, intrathecal,intraventricular, intramuscular, intrahepatic, intradermal,subcutaneous, oral (e.g., inhalation, buccal, sublingual, intranasal),transdermal (topical), transmucosal, and rectal administration. Nucleicacids can be delivered using electrical or magnetic stimulation, ordirect physical uptake using hydrodynamic pressure. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Gene therapy compositions suitable for injectable use can includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, in certain embodiments,carriers can include physiological saline, bacteriostatic water,Cremophor EL® (BASF, Parsippany, N.J.) or phosphate buffered saline(PBS). However, the art as relates to a specific viral delivery vectorwill be known to the skilled artisan and will provide appropriateconstituents for a gene therapy vector composition. A composition forinjection must be sterile (apart from the AAV or other viral vectoremployed for delivery) and should be fluid to the extent that easysyringability exists. In certain embodiments, such compositions arestable under the conditions of manufacture and storage and are preservedagainst the contaminating action of microorganisms such as bacteria andfungi. Exemplary carriers can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the genetherapy vectors disclosed herein in the required amount in a selectedsolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehicle,which contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying which yieldsa powder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

High titer AAV preparations can be produced using techniques known inthe art, e.g., as described in U.S. Pat. No. 5,658,776 and Viral Vectorsfor Gene Therapy: Methods and Protocols, ed. Machida, Humana Press,2003.

For administration by inhalation, gene delivery compositions can bedelivered in the form of an aerosol spray from pressured container ordispenser which contains a suitable propellant, e.g., a gas such ascarbon dioxide, or a nebulizer. Such methods include those described inU.S. Pat. No. 6,468,798, which is incorporated herein by reference.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compositions lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For a compositionsused in the method of the invention, the therapeutically effective dosecan be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compositions which achieves a half-maximal inhibition ofsymptoms) as determined in cell culture. Such information can be used tomore accurately determine useful doses in humans. Levels in plasma maybe measured, for example, by high performance liquid chromatography.

Expression constructs of the invention can be delivered to a subject by,for example, inhalation, orally, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994), Proc. Natl. Acad. Sci. USA, 91,3054-3057) or by any aforementioned delivery route. The pharmaceuticalpreparation of the delivery vector can include the vector in anacceptable diluent, or can comprise a slow release matrix in which thedelivery vehicle is imbedded. Alternatively, where the complete deliveryvector can be produced intact from recombinant cells, e.g., retroviralvectors, the pharmaceutical preparation can include one or more cellswhich produce the gene delivery system.

The expression constructs may be constructs suitable for use in theappropriate expression system and include, but are not limited toretroviral vectors, linear expression cassettes, modified mRNAs,plasmids and viral or virally-derived vectors, as known in the art. Thenucleic acids can be modified covalently, such as peptide nucleic acidsor base modified ribonucleic acids. Such expression constructs mayinclude one or more promoters as detailed elsewhere herein.

Suitable amounts of a gene therapy composition must be introduced andthese amounts can be empirically determined using standard methods.

The gene therapy composition can be formulated as a composition whichcomprises a pharmacologically effective amount of a transgene and/orviral vector containing a transgene, and pharmaceutically acceptablecarrier. A pharmacologically or therapeutically effective amount refersto that amount of gene therapy agent effective to produce the intendedpharmacological, therapeutic or preventive result. The phrases“pharmacologically effective amount” and “therapeutically effectiveamount” or simply “effective amount” refer to that amount of a genetherapy transgene effective to produce the intended pharmacological,therapeutic or preventive result. For example, if a given clinicaltreatment is considered effective when there is at least a 20% increasein a measurable parameter associated with a disease or disorder, atherapeutically effective amount of a gene therapy composition for thetreatment of that disease or disorder is the amount necessary to effectat least a 20% increase in that parameter. In another example, if agiven clinical treatment is considered effective when there is at least10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% or more increase in ameasurable parameter associated with a disease or disorder, atherapeutically effective amount of a gene therapy composition for thetreatment of that disease or disorder is the amount necessary to effectat least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% or more increase inthat parameter.

Markers of Transgene Expression/Activity

Toxicity and therapeutic efficacy of gene delivery compositions can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Gene therapy compositions which exhibit high therapeuticindices are preferred. While gene therapy compositions that exhibittoxic side effects may be used, care should be taken to design adelivery system that targets such compositions to the site of affectedtissue in order to minimize potential damage to uninfected cells and,thereby, reduce side effects.

In certain embodiments, membrane localization, including intracellularlocalization of a transgene or product thereof, e.g., NPC1, is assessedin the subject and/or in cells of the subject. In other embodiments,assessment of the efficacy of NPC1 transgene delivery is performed viameasurement of cholesterol uptake (e.g., endocytic cholesterol uptake)of the cells of a subject and/or via phenotypic assessment of a subjectbefore and after administration of the AAV-NPC composition(s). Suchassessment can be performed within days of administration of an AAV-NPCcomposition of the invention, or can be performed at a time of, e.g.,one week, two weeks, three weeks, one month, two months, three months,four months, five months, six months, one year or morepost-administration. The use of previously described biomarkers such asunesterified cholesterol, sphingomyelin, bis(monoacylglycero)phosphate,glucosylceramide, lactosylceramide, globotriaosylceramide, freesphingosine, gangliosides GM2 and GM3; galectin-3 (LGALS3) apro-inflammatory molecule, and cathepsin D (CTSD), a lysosomal asparticprotease; and cholesterol oxidation products and neurosteroids such ascholestane-3β,5α, 6β-triol (‘triol’), a cholesterol oxidation productthat is elevated 10-fold in the plasma of NPC1 subjects, and24(S)-hydroxycholesterol (24(S)-HC), an enzymatically generatedoxygenated cholesterol that is reduced in the plasma of NPC1 subjects.Untargeted metabolomics are likewise envisioned to monitor the efficiacyand activity of AAV gene therapy for NPC.

In certain embodiments, prior to treatment, a subject is assessed forthe identity of genetic deficiency that has produced NPC in thesubject—whether NPC1 or NPC2- and the subject is then administered anappropriate NPC1 or NPC2 transgene depending upon the outcome of suchassessment. In another embodiment, the transgene encodes a transgenethat has been codon optimized for human expression designated coNPC1 orcoNPC2. Methods for diagnosing NP disease can be found, for example, inU.S. Pat. Nos. 4,039,388, 5,686,240, 6,426,198, and 7,045,675 each ofwhich are incorporated by reference.

The invention further provides a method to treat related disorders ofunesterified cholesterol accumulation, such as atherosclosis.

The level or activity of a transgene mRNA or polypeptide can bedetermined by a suitable method now known in the art or that is laterdeveloped, e.g., analyzing expression levels by PCR, hybridization,microarrays, or other similar methodologies. Suitable primers, probes,and oligonucleotides capable of performing such detection will be knownand readily obtainable in the art. It can be appreciated that the methodused to measure a transgenic mRNA and/or the expression of a transgenicprotein can depend upon the nature of the transgene. Such measurementscan be made on cells, cell extracts, tissues, tissue extracts or othersuitable source material.

The determination of whether the expression of a transgene has beenincreased can be by a suitable method that can reliably detect changesin RNA or protein levels. In certain embodiments, the determination ismade by introducing into the environment of a cell a gene therapycomposition of the invention such that at least a portion of the genetherapy vector enters the cytoplasm (optionally, the nucleus;optionally, with nuclear chromosomal integration), and then measuringthe level of the transgene RNA and/or polypeptide. The same measurementis made on identical untreated cells and the results obtained from eachmeasurement are compared.

Combination Therapies

It is contemplated that the compositions of the current invention can becombined with other proposed therapies (e.g., for NPC) to slow diseaseprogression and ameliorate symptoms, even in patients with advanceddisease. There are no published standards of care for NPC other thansymptomatic treatment of disease manifestations—seizures are controlledas possible and supportive care is provided as needed. In oneembodiment. AAV gene therapy would be combined with the pharmaceuticalexcipient 2-hydroxypropyl-β-cyclodextrin (HPβCD). In another, AAV genetherapy would be combined therapies shown to have modest efficacy inmouse models or cell culture studies including treatment withantioxidants such as N-acetylcysteine; vitamin E or derivatives such asα-tocepherol or δ-tocepherol; miglustat, a small imino sugar thatpartially inhibits glucosylceramide synthase and the synthesis of allglucosylceramide-based glycosphingolipids; curcumin to compensates forthe lysosomal calcium defect by elevating cytosolic calcium; thenon-steroidal anti-inflammatory drug ibuprofen or related compounds toreduce central nervous system inflammation; donepezil, a widely usedacetylcholinesterase (AChE) inhibitor; or Histone deacetylase inhibitors(HDACi) such as vorinostat. In another embodiment, AAV gene therapywould be combined with other therapies that have a theoretical basis forefficiacy—such as those that influence cholesterol metabolism, but havelimited efficacy to date. These include the cholesterol-lowering agentscholestyramine, lovastatin, and nicotinic acid as well as alow-cholesterol diet

Dosage

Human dosage amounts can initially be determined by extrapolating fromthe amount of compound used in mice, as a skilled artisan recognizes itis routine in the art to modify the dosage for humans compared to animalmodels. In certain embodiments it is envisioned that the dosage may varyfrom between about 1 mg compound/Kg body weight to about 5000 mgcompound/Kg body weight; or from about 5 mg/Kg body weight to about 4000mg/Kg body weight or from about 10 mg/Kg body weight to about 3000 mg/Kgbody weight; or from about 50 mg/Kg body weight to about 2000 mg/Kg bodyweight; or from about 100 mg/Kg body weight to about 1000 mg/Kg bodyweight; or from about 150 mg/Kg body weight to about 500 mg/Kg bodyweight. In other embodiments this dose may be about 1, 5, 10, 25, 50,75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000,4500, 5000 mg/Kg body weight. In other embodiments, it is envisaged thathigher does may be used, such doses may be in the range of about 5 mgcompound/Kg body to about 20 mg compound/Kg body. In other embodimentsthe doses may be about 8, 10, 12, 14, 16 or 18 mg/Kg body weight. Ofcourse, this dosage amount may be adjusted upward or downward, as isroutinely done in such treatment protocols, depending on the results ofthe initial clinical trials and the needs of a particular patient.

In certain embodiments, a suitable dosage unit of a transgene vector isin the range of 0.001 to 0.25 milligrams per kilogram body weight of therecipient per day, or in the range of 0.01 to 20 micrograms per kilogrambody weight per day, or in the range of 0.001 to 5 micrograms perkilogram of body weight per day, or in the range of 1 to 500 nanogramsper kilogram of body weight per day, or in the range of 0.01 to 10micrograms per kilogram body weight per day, or in the range of 0.10 to5 micrograms per kilogram body weight per day, or in the range of 0.1 to2.5 micrograms per kilogram body weight per day. A gene therapycomposition comprising the transgene can be administered once or onmultiple occasions.

Data can be obtained from cell culture assays and animal studies toformulate a suitable dosage range for humans. The dosage of compositionsof the invention can lie within a range of circulating concentrationsthat include the ED₅₀ (as determined by known methods) with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For acomposition used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the composition that includes the IC₅₀ (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsof a gene therapy composition in plasma may be measured by standardmethods, for example, by high performance liquid chromatography.

In certain embodiments, the dosage may be in terms of vectorconcentration. For example, the concentration of gene therapy vectordescribed herein is at least: (a) 5, 6, 7, 8, 8.4, 9, 9.3, 10, 15, 20,25, or 50×10¹² gc/ml; (b) 5, 6, 7, 8, 8.4, 9, 9.3, 10, 15, 20, 25, or50×10⁹ tu/ml (“transducing units per ml”); (c) 5, 6, 7, 8, 8.4, 9, 9.3,10, 15, 20, 25, or 50×10¹⁰ iu/ml (“international units per ml”), or (d)5, 6, 7, 8, 8.4, 9, 9.3, 10, 15, 20, 25, or 50×10¹⁰ pfu/ml (“plaqueforming units per ml”).

Kits and/or Pharmaceutical Packages

The gene therapy compositions of the invention can be included in a kitand/or pharmaceutical package, container, pack, or dispenser togetherwith instructions for administration.

The disclosure provides kits for the treatment or prevention of disease,e.g., NP disease, Type C. In one embodiment, the kit includes atherapeutic or prophylactic composition containing an effective amountof an agent of the invention (e.g., NPs) in unit dosage form. In someembodiments, the kit comprises a sterile container which contains atherapeutic or prophylactic compound; such containers can be boxes,ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or othersuitable container forms known in the art. Such containers can be madeof plastic, glass, laminated paper, metal foil, or other materialssuitable for holding medicaments.

If desired an agent of the disclosure is provided together withinstructions for administering it to a subject having or at risk ofdeveloping a disease. The instructions will generally includeinformation about the use of the composition for the treatment orprevention of the disease (e.g., NPC). In other embodiments, theinstructions include at least one of the following: description of thecompound; dosage schedule and administration for treatment or preventionof the disease or symptoms thereof; precautions; warnings; indications;counter-indications; overdosage information; adverse reactions; animalpharmacology; clinical studies; and/or references. The instructions maybe printed directly on the container (when present), or as a labelapplied to the container, or as a separate sheet, pamphlet, card, orfolder supplied in or with the container.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, immunology, cell biology, cellculture and transgenic biology, which are within the skill of the art.See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989,Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rdEd. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.);Ausubel et al., 1992), Current Protocols in Molecular Biology (JohnWiley & Sons, including periodic updates); Glover, 1985, DNA Cloning(IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow andLane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Jakoby and Pastan, 1979; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney. Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987. Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6thEdition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986); Westerfield, M., The zebrafish book. Aguide for the laboratory use of zebrafish (Danio rerio), (4th Ed., Univ.of Oregon Press, Eugene, 2000).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention.

EXAMPLES

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

Example 1. Neuron-Specific Adeno-Associated Viral (AAV) Vector for NPC1Delivery

To develop a new class of gene-based therapeutics for NPC in humans, aseries of adeno-associated viral (AAV) vectors were developed fordelivery of the NPC1 gene, first to neurons and then to other celltypes. An NPC1 gene was engineered for expression under the control ofthe calmodulin promoter. This expression cassette was cloned into anempty AAV2 vector, which created AAV-_(mini)calmodulin-NPC1. This vectorwas encapsidated with an AAV serotype 9 capsid and used to produceAAV2/9 _(mini)calmodulin-NPC.

The pre-clinical efficacy of AAV-_(mini)calmodulin-NPC as a treatmentfor NPC was accomplished in vivo. Npc1^(−/−) mice (n=9) received 1×10¹²GC of AAV9-_(mini)CaMKII-NPC1 or an equivalent reporter control,AAV9-_(mini)CaMKII-GFP (n=6), between 20 and 25 days of life deliveredby retroorbital injection. To achieve neuronal transduction, we reliedupon the well-established property of AAV9 vectors to cross theblood-brain barrier and transduce neurons after systemic delivery.Relative to the untreated or AAV-GFP treated Npc1^(−/−) mice [meansurvival 66 days], the Npc1^(−/−) mice that receivedAAV9-_(mini)CaMKII-NPC1 exhibited an increased life span (mean survival105 days; P<0.02) Systemic delivery by retroorbital injection of AAV2/9_(mini)calmodulin-NPC into Npc1^(−/−) mice resulted in increasedsurvival and mitigation of disease related symptoms. These resultsestablished this AAV as a successful gene therapeutic for NPC.

Example 2. Adeno-Associated Viral (AAV) Vector for NPC1 Delivery andUbiquitous Expression

A similar vector expressing the NPC1 gene from the elongation factor 1 α(EF1α) promoter is also synthesized.

While AAV2/9 _(mini)calmodulin-NPC directed the expression of NPC inneurons, the AAV2/9-_(mini)EF1α-NPC is expected to produce morewidespread expression, because the elongation factor 1 α promoter isknown to direct expression in all cell types (ubiquitous). This genetherapy vector could be used to treat neurological, hepatic and otherextraCNS symptoms in NPC patients. A truncated promoter was used tofirst create and AAV that expressed eGFP as a control. This AAV iscalled AAV-_(mini)EF1α-eGFP. Next, eGFP was excised, the WPRE elementwas removed both were replaced with the human NPC1 cDNA to createAAV-_(mini)EF1α-NPC1 and used to treat Npc1^(−/−) mice.

Example 3. Therapeutic Treatment of NPC Subjects Using Adeno-AssociatedViral (AAV) Vector for NPC1 Neuron-Specific Delivery

Human NPC subjects are identified via methods known in the art,optionally including genetic testing of NPC1 and/or NPC2 loci to confirmphenotype-based diagnoses. NPC subjects are administeredAAV-_(mini)calmodulin-NPC (optionally, AAV2/9 _(mini)calmodulin-NPC) byinjection as a treatment for NPC. Other AAV serotypes will be used, suchas AAVrh8 and AAV rh10. Following injection, one or more NPC-associatedphenotypes (e.g., seizure incidence) and biomarkers (e.g., gangliosides,unesterified cholesterol) are assessed in treated NPC subjects and/orthe level(s) of NPC1 or NPC2 mRNA or polypeptide expression is measuredin the cells of NPC subjects, and is compared to a suitable control(e.g., baseline measurements pre-treatment, a control population of NPCsubjects, etc.). The in vivo therapeutic efficacy of gene therapytreatment employing AAV and the AAV-_(mini)calmodulin-NPC vector of theinvention is thereby identified.

Example 4. Therapeutic Treatment of NPC Subjects NPC1 Delivery andUbiquitous Expression

Human NPC subjects are identified via methods known in the art,optionally including genetic testing of NPC1 and/or NPC2 loci to confirmphenotype-based diagnoses. NPC subjects are administeredAAV-_(mini)EF1α-NPC1 (optionally, AAV2/9 AAV-_(mini)EF1α-NPC1) byinjection as a treatment for NPC. Other AAV serotypes will be used, suchas AAVrh8 and AAV rh10. Following injection, one or more NPC-associatedphenotypes (e.g., seizure incidence) and biomarkers (e.g., gangliosides,unesterified cholesterol, measures of hepatic function) are assessed intreated NPC subjects and/or the level(s) of NPC1 or NPC2 mRNA orpolypeptide expression is measured in the cells of NPC subjects, and iscompared to a suitable control (e.g., baseline measurementspre-treatment, a control population of NPC subjects, etc.). The in vivotherapeutic efficacy of gene therapy treatment employing AAV and theAAV-_(mini)EF1α-NPC1 vector of the invention is thereby identified.

Example 5. AAV9-_(mini)CaMKII-GFP Mediated Transduction of NeuronalPopulations in Npc1^(−/−) Mice

An AAV vector that could both transduce and express human NPC1 gene inneurons was designed using a small neuro-specific promoter, CaMKII (375bp). CaMKII was selected because it could be cloned into an AAV vectorwith NPC1 cDNA (3.8 kb) given the size limitation for AAV packaging. Thefinal size of AAV vector including the 5′ and 3′ ITRs, which flanked theCaMKII promoter, NPC1 cDNA, and a polyadenylation signal, was around 4.8kilobases. This vector construct was packaged using a serotype 9 capsid,which has been shown to be able to cross the blood brain barrier aftersystemic delivery and to be highly effective at transducing neurons. Inorder to define the tropism and expression pattern of AAV9-_(mini)CaMKIIvector in vivo, a vector expressing the reporter, green fluorescent(AAV9-_(mini)CaMKII-GFP) was used. A single retro-orbital injection of1×10¹² GC of AAV9-_(mini)CaMKII-GFP was performed into Npc1^(−/−) miceat day of life 23. The expression pattern of the endogenous CaMKIIpromoter in an adult wildtype mouse is shown in FIG. 16a .Immunohistochemical imaging of the AAV9-_(mini)CaMKII-GFP treatedNpc1^(−/−) mice taken at 9 weeks of age showed a remarkably similarexpression pattern, with strong GFP expression in the olfactory bulb,cerebral cortex, striatum and hippocampus, with weaker GFP expressionthroughout the midbrain and hindbrain (FIG. 16b ).

In contrast to the endogenous expression pattern, little cerebellarincorporation was observed (FIG. 16b ). All GFP-positive cells were alsolabeled with NeuN, indicating that expression of the viral gene productonly occurred in neurons (FIG. 16c ). Higher magnification images of thecerebral cortex (FIG. 16d, e ) and the CA3 field of the hippocampus(FIG. 16f, g ) clearly show the neurospecific double-labeling(arrowheads) and neuronal morphology of the AAV9-_(mini)CaMKII-GFPtransduced cells.

Example 6 AAV9-_(mini)CaMKII-NPC1 Gene Delivery Improves Survival andDelays Loss of Motor Function and Weight Decline of Npc1^(−/−) Mice

To test the efficacy of gene therapy as treatment for NPC at differentdelivery time points, Npc1^(−/−) pups (n=6) received 2×10¹¹ GC ofAAV9-_(mini)CaMKII-NPC1 between 1 and 3 days and Npc1^(−/−) mice (n=9)received 1×10¹² GC of AAV9-_(mini)CaMKII-NPC1 between 20 and 25 days oflife by retroorbital injection. A control group of Npc1^(−/−) mice (n=6)received 1×10¹² GC of AAV9-_(mini)CaMKII-GFP between 20 and 25 days oflife delivered by retroorbital injection. Consistent with previousreports, the untreated Npc1^(−/−) mice (n=16) had a mean survival of 69days and AAV9-_(mini)CaMKII-GFP treatment of Npc1^(−/−) mice had noeffect on survival with a mean survival of 65 days (FIGS. 17a and b ).In contrast, Npc1^(−/−) mice that received AAV9-_(mini)CaMKII-NPC1 ateither 1-3 or 20-25 days of life exhibited an increased life span, witha mean survival of 97 and 103 days, respectively (P<0.001, FIGS. 17a andb ). Relative to the untreated Npc1^(−/−) mice, age matchedAAV9-_(mini)CaMKII-NPC1 treated Npc1^(−/−) mice at 8-9 weeks of lifedisplayed physiological improvements that corresponded to an objectiveimprovement in motor function, where mice appeared to maintain theirstrength and coordination to walk and explore the home-cage with reducedsigns of tremor.

While gene delivery improved both survival and mobility of Npc1^(−/−)mice, it had no significant effect on mass between 4 and 9 weeks. Theweek at which the Npc1^(−/−) mice achieved their maximal or peak weightwas used to determine if gene delivery delayed or prevent the weightloss that occurs in untreated Npc1^(−/−) (FIG. 17c ). Both untreated(n=16) and AAV9-_(mini)CaMKII-GFP treated (n=6) mice almost uniformlyreach their peak weights at 6 weeks. While Npc1^(−/−) pups (n=6) thatreceived 2×10¹¹ GC of AAV9-_(mini)CaMKII-NPC1 between 1 and 3 days andNpc1^(−/−) mice (n=9) that received 1×10¹² GC of AAV9-_(mini)CaMKII-NPC1between 20 and 25 days of life on average reach their peak weight at 8weeks (relative to untreated Npc1^(−/−) mice P<0.01 and P=0.08,respectively). Because the weight decline of untreated mice began at 6weeks and most untreated mice did not survive beyond 9 weeks, Thepercentage weight change from 6 to 9 weeks (% weight (wt)change=[wt_(9weeks)−wt_(6weeks)]/wt_(6weeks)×100) of untreated,AAV9-_(mini)CaMKII-NPC1 and AAV9-_(mini)CaMKII-GFP treated Npc1^(−/−)mice (FIG. 17d ) was compared. Untreated and AAV9-_(mini)CaMKII-GFPtreated mice that had a percent weight change of −17% and −23%,respectively. Relative to untreated Npc1^(−/−) mice,AAV9-_(mini)CaMKII-NPC1 Npc1^(−/−) pups treated at 1-3 days and micetreated at 20-25 days demonstrated a significant reduction in weightloss, −3.8% (P<0.02) and −2.3% (P<0.001), respectively.

Example 7. AAV9-_(mini)CaMKII-NPC1 Treatment Increases NPC1 ProteinExpression and Reduces Intracellular Accumulation of Cholesterol inDisease-Affected Brain Regions

Layer V of the cerebral cortex (LV) and the CA3 pyramidal layer of thehippocampus (CA3) were chosen as ideal brain structures to assess theeffectiveness of AAV9-_(mini)CaMKII-NPC1 administration. The pyramidalneurons in these regions are prone to high levels of unesterifiedcholesterol accumulation, but show no evidence of neuronal death in thismouse model, occluding cell loss as a confounding factor in ouranalysis. Retro-orbital administration of AAV9-_(mini)CaMKII-NPC1 wasperformed on Npc1^(−/−) mice at day 23, with PBS-control injections madein corresponding Npc1^(−/−) and Npc1^(+/+) animals. Immunohistochemicalassessment of the resulting Npc1^(+/+) brain tissue at 9-weeks of age(FIG. 18a-f ) showed the stereotypical pattern of Npc1 proteinexpression and cholesterol localization. Npc1 staining was noted in NeuNpositive neurons throughout the forebrain with no major intracellularaccumulations of unesterified cholesterol observed by filipin staining,including the neocortical and hippocampal regions of interest. (L-Vneurons; Npc1=12.04 mpi, filipin=5.84 mpi, FIG. 18c, d )(CA3 neurons;Npc1=10.63 mpi, filipin=2.05 mpi, FIG. 18e, f ). The majority of thefilipin signal in Npc1^(+/+) mice was found in myelin-rich structuressuch as the corpus calosum. The reverse was true in the PBS injectedNpc1^(−/−) mice (FIG. 18g-l ). Almost no specific staining was detectedwith the Npc1 antibody, and many of the NeuN positive neurons throughoutthe forebrain exhibited the high levels of unesterified cholesterolaccumulation typical of late-stage NPC disease pathology, including theLV (Npc1=2.98 mpi, filipin=28.9 mpi, FIG. 3i, j ) and CA3 (Npc1=1.54mpi, filipin=33.80 mpi, FIG. 3k, l ) neurons.

Analysis of the AAV9-_(mini)CaMKII-NPC1 treated Npc1^(−/−) mice (FIG.18m-r ) revealed an intermediate phenotype. While intracellularcholesterol accumulation was widespread throughout the brain, theaverage neuronal intracellular filipin intensity was 21.01 mpi in LV(FIG. 18o, p ) and 21.33 mpi in CA3 (FIG. 18q, r ), significantly lowerthan observed in the PBS-control Npc1^(−/−) mice (P<0.05 and P<0.01,respectively). This coincided with an NPC1 signal inAAV9-_(mini)CaMKII-NPC1 treated Npc1^(−/−) neurons of 9.76 mpi in LV(FIG. 18o, p ) and 8.60 mpi in CA3 (FIG. 18q, r ). While clearly belowNpc1^(+/+) neuron levels, this still represents a significant increaseof NPC1 protein in AAV9-_(mini)CaMKII-NPC1 treated Npc1^(−/−) mice whencompared to the Npc1^(−/−) vehicle controls (P<0.01 and P<0.0001respectively, all quantifications in FIG. 18s-v ).

Example 8. Biochemical Correction of Npc1^(−/−) Neurons Following GeneDelivery

Having identified that the AAV9-_(mini)CaMKII-NPC1 transducedNPC1-affected brain regions, induced production of NPC1 protein andreduced cholesterol pathology, it was determined whether theAAV-mediated expression of NPC1 was biochemically functional in aphysiologically typical manner in Npc1^(−/−) mice treated with AAV9 atday 23. Close inspection of the LV neuronal population inAAV9-_(mini)CaMKII-NPC1 treated mice revealed a punctuate andperinuclear intracellular localization pattern for NPC1 protein, typicalof a lysosomal distribution. In addition, LV neurons that lackedcholesterol accumulations were strongly NPC1-positive, while nearbyweakly- or non-transduced neurons remained filipin positive (FIG. 19a, b).

Plotting of NPC1 expression versus cholesterol accumulation of the LVneuron population analyzed in AAV9-_(mini)CaMKII-NPC1 treated miceagainst those of control Npc1^(+/+) and Npc1^(−/−) mice revealed that21% (±5.71 S.E.M, n=4) had been biochemically corrected to normal levels(FIG. 19c, d ). The same phenomenon was observed in the CA3 neurons,where 31% (±1.58 S.E.M, n=4) of this population inAAV9-_(mini)CaMKII-NPC1 treated mice was indistinguishable from normal,healthy neurons (FIG. 19e-h ).

Comparison of the percentage of successfully transduced neurons in theAAV9-_(mini)CaMKII-GFP treated Npc1^(−/−) mice (FIG. 19i, j ) and thepercentage of biochemically-corrected neurons in theAAV9-_(mini)CaMKII-NPC1 treated Npc1^(−/−) mice (FIG. 19k, l ) was madeto assess whether the levels of biochemical correction directly matchedthe transduction pattern of the AAV-_(mini)CaMKII-GFP construct.Penetrance of the AAV9-_(mini)CaMKII-GFP was 14.9% (±0.60 S.E.M, n=3) inLV neurons, and 27.7% (±0.75 S.E.M, n=3) in CA3 neurons, notsignificantly different to that of the AAV9-_(mini)CaMKII-NPC1(quantification in FIG. 19m, n ), indicating the level of biochemicalcorrection observed in Npc1^(−/−) neurons corresponded to transductionpattern of the AAV9-_(mini)CaMKII-GFP construct.

Example 9. Delayed Purkinje Neuron Loss in Npc1^(−/−) Mice Treated withAAV9-_(mini)CaMKII-NPC1

Significant improvements in lifespan and physiological criteria areusually accompanied by a preservation of cerebellar Purkinje cells inNPC1. Following immunohistochemical evaluation of Purkinje cell numbersat 9 weeks of age in our experimental groups, a significant delay in thetypical anterior-to-posterior loss of these neurons uponAAV9-_(mini)CaMKII-NPC1 treatment (FIG. 20a-e ) was noticed. In theNpc1^(+/+) control mice, Purkinje cell numbers remained at normal levels(31.01, 26.94, and 29.51 cells/mm of pcl (Purkinje cells per mm ofPurkinje cell layer:granule cell layer interface) in lobules VI, VII andIX respectively), but large-scale neuron loss was observed in Npc1^(−/−)control mice (2.435, 3.523, and 9.469 cells/mm of pcl in lobules VI, VIIand IX respectively). While Purkinje cell loss had initiated in anteriorlobules I-V in AAV9-_(mini)CaMKII-NPC1 treated Npc1^(−/−) mice,significantly more neurons were still present when compared to theNpc1^(−/−) control mice, with 9.374 cells/mm of pcl in lobule VI(P<0.05), 9.716 cells/mm of pcl in lobule VII (P<0.05), and 17.22cells/mm of pcl in lobule IX (P<0.01)(quantifications in FIG. 20f-h ),suggestive of an indirect AAV9-_(mini)CaMKII-NPC1 gene therapy-mediateddelay in Purkinje cell death and motor function decline.

Example 10. AAV9-_(mini)EF1α-NPC1 Treatment Significantly ImprovesSurvival and Increases Growth Relative to AAV9-_(mini)CaMKII-NPC1Treatment (FIG. 21)

AAV9 vector that utilized a ubiquitous promoter was designed todetermine whether AAV gene therapy used to correct both neuronal as wellas other cell types in the brain and other organs might improve upon theefficacy that was observed with AAV9-_(mini)CaMKII-NPC1 gene delivery.The _(mini)CaMKII promoter was replaced with a truncated EF1α promoter(227 bp). The final size of AAV vector including the 5′ and 3′ ITRs,which flanked the EF1α promoter, NPC1 cDNA, and a polyadenylationsignal, was 4.7 kilobases.

Npc1^(−/−) mice (n=8) received 1×10¹² GC of AAV9-_(mini)EF1α-NPC1 at 24days of life by retroorbital injection. Relative to Npc1^(−/−) mice(n=9) treated with 1×10¹² GC of AAV9-_(mini)CaMKII-NPC1 between 20-25days of life by retroorbital injection and untreated Npc1^(−/−) mice(n=16) with a mean survival of 97 and 69 days, respectively, theAAV9-_(mini)EF1α-NPC1 treated Npc1^(−/−) mice (n=8) had a significantincrease (P<0.01) in survival with a mean survival >121 days (FIG. 23).Relative to the untreated Npc1^(−/−) mice, age matchedAAV9-_(mini)EF1α-NPC1 treated Npc1^(−/−) mice at 8-9 weeks of lifedisplayed physiological improvements that corresponded to an objectiveimprovement in motor function, where mice appeared to maintain theirstrength and coordination to walk and explore the home-cage with reducedsigns of tremor.

The AAV9-_(mini)EF1α-NPC1 treated Npc1^(−/−) mice reached their meanpeak weight at week 12, which was significantly later (P=0.02) thanNpc1^(−/−) mice treated with AAV9-CaMKII-NPC1 that reached their meanpeak weight at week 8 (FIG. 22a ). The mean percentage weight gainbetween 6 and 9 weeks for AAV9-_(mini)EF1α-NPC1 treated Npc1^(−/−) micewas 8.8% and was greater than the mean percentage weight change forAAV9-_(mini)CaMKII-NPC1 treated Npc1^(−/−) mice, which was −2.3%, butthis difference was not significant (FIG. 22b ).

Discussion

The studies were done to test the efficacy of AAV mediated gene deliveryas a treatment for NPC1, a progressive and lethal neurological disease.AAV9 was selected for gene delivery because it has been shown to be ableto cross the blood brain barrier and transduce neurons and glial cells,used successfully in many other murine models of inherited neurologicaldiseases, and is being tested in human clinical to treat other inheritneurological diseases. The neuro-specific CaMKII promoter was selectedfirst because of its small size and ability to express in Purkinjecells, the death of this class of neuron is thought to lead to the motorloss seen in NPC1 disease. AAV9-_(mini)CaMKII-NPC1 was delivered in theneonatal period and at 3 weeks of life to determine if treating theNpc1^(−/−) mice early would increase the efficacy of AAV treatment.

Npc1^(−/−) neonates and mice treated with AAV9-_(mini)CaMKII-NPC1 had asignificant increased survival, superior motor activity at 9 weeks anddelayed weight loss relative to untreated and AAV9-_(mini)CaMKII-GFPtreated Npc1^(−/−) mice. No significant difference in efficacy wasobserved regardless of the timing of the AAV9 delivery. The delayeddelivery of AAV9 more accurately reflects the timing of AAV9 delivery ina potential human clinical trial as NPC1 disease is not typicallydiagnosed in the neonatal period.

Immunohistochemistry for NPC1/Npc1 and filipin staining confirmed that asubset of neurons of the AAV9 treated Npc1^(−/−) mice expressed NPC1 andshowed a corresponding reduction of filipin staining relative untreatedNpc1^(−/−) mice at 9 weeks. These results demonstrate a correlationbetween expression of NPC1 after gene deliver, a reduction incholesterol storage, and an improvement of the NPC1 disease phenotype.However, substantially fewer transduced Purkinje neurons were detectedin the cerebellum, an area where Purkinje neuron loss parallels theprogression of the NPC1 disease in mouse model. The low transduction inthe cerebellum may explain why AAV9-_(mini)CaMKII-NPC1 gene deliveryonly delayed the progression of the disease in the murine model of NPC1and why mice did not achieve a normal life expectancy or the same weightas wild-type mice. Despite the lower AAV9 transduction in thecerebellum, the AAV9 treated mice had greater cerebellar Purkinje neuronsurvival at 9 weeks relative to untreated Npc1^(−/−) mice.

Because the AAV9-_(mini)CaMKII-NPC1-treated Npc1^(−/−) mice onlyachieved a modest increase in life expectancy, the vector wasreengineered to express the NPC1 gene using a ubiquitous EF1α promoterin an attempt to improve the efficacy of the gene therapy that wasinitially observed. Npc1^(−/−) mice treated with the new vector,AAV9-_(mini)EF1α-NPC1, shows a significant increase in life expectancyand growth relative to Npc1^(−/−) mice treated withAAV9-_(mini)CaMKII-NPC. Since it utilized the same AAV9 serotype todeliver both vector constructs, the only difference between the twovectors are the cells types in which NPC1 is being expressed. Thisresult suggests that correction of non-neuronal cells in the brain suchas glial cells and/or cells types outside the central nervous system byAAV9-_(mini)EF1α-NPC1 is responsible for the increased therapeuticeffect that was observed. More experiments will be need to determine theunder lying differences in efficacy between these two AAV vectors andunderstanding these differences could lead to a further improvement ofAAV gene therapy for NPC disease.

Currently, the Npc1^(−/−) mice treated with AAV9-_(mini)EF1α-NPC1 arealive and appear relatively healthy, although their weights are lowerthan their wild-type littermates. Long-term monitoring of theseAAV9-_(mini)EF1α-NPC1 treated mice will be required to determine howlong the therapeutic benefit of AAV gene therapy will persist, but theseresults are extremely promising and further vector redesign may not beneeded to justify advancement to clinical trials. However, furtherrefinement of route of administration, promoter usage and/or combinationwith other interventions (such as 2-hydroxypropyl-β-cyclodextrin orHDACi) could potentially improve the effectiveness of AAV gene therapyas a treatment for NPC. These studies are first to demonstratepre-clinical efficacy of AAV gene therapy as a therapeutic approach forNPC disease.

Materials and Methods Regarding Examples 5-10:

AAV Vector Design and Production.

The expression vector, pENN.AAV. _(mini)CaMKII0.4.eGFP.rBG (PL-C-PV1474) was obtained from the University of Pennsylvania Vector Core. Thisvector contains transcriptional control elements from the, mousecalcium/calmodulin-dependent protein kinase II (CaMKII) promoter,cloning sites for the insertion of a complementary DNA, and the rabbitβ-globin polyA signal. Terminal repeats from AAV serotype 2 flank theexpression cassette. The eGPF cDNA was excised frompENN.AAV._(mini)CaMKII0.4.eGFP.rB plasmid and replaced with the humanNPC1 cDNA. This newly created vector was called AAV-_(mini)CaMKII-NPC1.These AAV vectors were packaged into an AAV9 capsid, purified by cesiumchloride centrifugation, and titered by qPCR as previously described.The _(mini)CaMKII promoter was removed from the AAV-_(mini)CaMKII-NPC1vector and replaced with a truncated EF1α (_(mini)EF1α) promoter. Thetruncated EF1α promoter was cloned into an eGFP expression vector andtransfected into 293T cells to test for promoter activity. The truncatedEF1α expressed GFP in 293T cells at levels similar to the full lengthEF1α promoter. The AAV2/9-_(mini)CaMKII-NPC1 and AAV29-_(mini)EF1α-NPC1vectors were produced by the Penn Vector Core at the University ofPennsylvania with published procedures.

Animals.

All animal work was done according to NIH-approved animal care and useprotocols. Heterozygous Npc1^(+/−) mice (BALB/c Nctr-Npc1^(mIN)/Jstrain) were bred to obtain control (Npc1^(+/+)) and mutant (Npc1^(−/−))littermates. Mice were weighed weekly and mutant mice were euthanized at9 weeks of age, typically the disease end-stage in our colony asdetermined by rapid weight loss and severe loss of motor function. Forevaluation of lifespan in AAV9-_(mini)CaMKII-NPC1 andAAV9-_(mini)CaMKII-GFP Npc1^(−/−) treated groups, end-stage wasdetermined using the same criteria.

Administration of AAV9.

Neonatal Npc1^(−/−) pups (1-3 days, n=6) received a retro-orbitalinjection of 2×10¹¹ GC of AAV9-_(mini)CaMKII-NPC1 in a total volume of10 μls. Npc1^(−/−) mice (20-25 days) received a retro-orbital injectionof 1×10¹² GC of AAV9-_(mini)CaMKII-GFP (n=6) virus orAAV9-_(mini)CaMKII-NPC1 (n=9) virus in a total volume of 50 μls.Alternatively, Npc1^(−/−) and Npc1^(+/+) received a sham injection of 50μls at 23 days of age.

Immunohistochemistry.

AAV9-_(mini)CaMKII-GFP treated Npc1^(−/−) mice (n=3),AAV9-_(mini)CaMKII-hNPC1 Npc1^(−/−) mice (n=4), control Npc1 mice (n=5)and control Npc1^(+/+) mice (n=3) were taken at 9 weeks of age forimmunohistochemical analysis. Mice were euthanized by CO₂ asphyxiationand transcardially perfused with 4% paraformaldehyde in phosphatebuffer. The brains were post-fixed for 24 h then cryoprotected in 30%sucrose until the tissues sank. Brains were then cryostat-sectionedparasagittally (25 μm) and floating sections collected in phosphatebuffered saline supplemented with 0.25% Triton-×100 (PBSt). Sectionswere incubated overnight at 4° C. with either rabbit anti-calbindin(1:3000, Swant), rabbit anti-Npc1 (1:2000), or mouse anti-NeuN (1:1000,MILIPORE) in PBSt, and the primaries detected using DyLight-488 goatanti-rabbit/mouse IgG or Alexa-594 anti-rabbit (1:1000 in PBSt, VectorLabs). Filipin (POLYSCIENCES INC.) staining was performed at finalconcentration of 50 μg/ml in PBSt. Sections were mounted andcoverslipped with ProLong Gold mounting medium (Life Technologies).

Image Analysis.

Images of the whole cerebellum or hippocampal/neocortical region weretaken using a Zeiss Axio Observer Z1 microscope fitted with an automatedscanning stage, Colibri II LED illumination and Zeiss ZEN software usinga high-res AxioCam MRm camera and a 20× objective. Each fluorophorechannel was pseudo-coloured in ZEN, exported as TIFF, and analyzed usingthe FIJI distribution of ImageJ, adjusting each channel for brightnessand contrast in an identical manner across all experimental groups.

For CA3 hippocampal and Layer V neocortical neuron analysis, the area ofa neuron was delineated according to the cell body size determined byNeuN staining and the mean pixel intensity (mpi) of the filipin and Npc1stain within the cell recorded. Every 5^(th) neuron was chosen at randomalong the CA3 or layer V axis to obtain a representative neuronalpopulation. 20 cells were counted in each region per section, and 3sections counted per brain (60 cells per n). To assess the level ofbiochemical correction in the AAV9-_(mini)CaMKII-NPC1 treated neuronalpopulation, gating boundaries were set at the lower 2% of the filipinand Npc1 intensities of the Npc1^(−/−) and Npc1^(+/+) control groups,respectively. Neurons with “lower than disease-baseline” levels ofcholesterol storage together with “above wildtype-baseline” levels ofNpc1 expression were considered biochemically corrected.

To analyze the transduction of the AAV9-_(mini)CaMKII-GFP virus, thetotal number of NeuN positive neurons was measured in a set area of CA3hippocampus or Layer V neocortex, and the % of those cellsdouble-labeled with GFP recorded (3 sections counted per brain, minimumof 72 cells counted in each section, total of 1086 hippocampal and 2367neocortical neurons measured).

Purkinje cells were counted by measuring the number of calbindinpositive Purkinje cell bodies with recognizable dendritic tree or axonalprojection still remaining within a given cerebellar lobule. Data wasexpressed as the number of Purkinje cells per mm of Purkinje celllayer:granule cell layer interface (pcl). The entire lobule was countedper section, with 3 sections counted per brain.

Statistical Analysis. Results are expressed as means±S.E.M, and analyzedfor statistical significance by ANOVA, where P<0.05 using Tukey'spost-test was considered significant. Kaplan-Meier survival curves weretested for significance using the Log-Rank Mantel-Cox test, whereresults were considered significant using a Bonferroni-correctedthreshold of P<0.0083 to account for multiple comparisons. Allstatistics were calculated using Graphpad Prizm software.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

INCORPORATION BY REFERENCE

Each of the applications and patents cited in this text, as well as eachdocument or reference cited in each of the applications and patents(including during the prosecution of each issued patent; “applicationcited documents”), and each of the PCT and foreign applications orpatents corresponding to and/or claiming priority from any of theseapplications and patents, and each of the documents cited or referencedin each of the application cited documents, are hereby expresslyincorporated herein by reference. More generally, documents orreferences are cited in this text, either in a Reference List before theclaims, or in the text itself; and, each of these documents orreferences (“herein-cited references”), as well as each document orreference cited in each of the herein-cited references (including anymanufacturer's specifications, instructions, etc.), is hereby expresslyincorporated herein by reference.

-   Reference for AAV Anc80: Zinn E. Pacouret S, Khaychuk V, Turunen H    T, Carvalho L S, Andres-Mateos E, Shah S, Shelke R, Maurer A C,    Plovie E, Xiao R, Vandenberghe L H. In Silico Reconstruction of the    Viral Evolutionary Lineage Yields a Potent Gene Therapy Vector. Cell    Rep. 2015 Aug. 11; 12(6):1056-68. doi: 10.1016/j.celrep.2015.07.019.    Epub 2015 Jul. 30. PubMed PMID: 26235624; PubMed Central PMCID:    PMC4536165.-   Reference for AAV PHP.B: Deverman B E, Pravdo P L, Simpson B P,    Kumar S R, Chan K Y, Banerjee A, Wu W L, Yang B, Huber N, Pasca S P,    Gradinaru V. Cre-dependent selection yields AAV variants for    widespread gene transfer to the adult brain. Nat Biotechnol. 2016    February; 34(2):204-9. doi: 10.1038/nbt.3440. Epub 2016 Feb. 1.    PubMed PMID: 26829320.

1. A nucleic acid construct comprising: (1) a viral vector sequence; and(2) an NPC1 gene sequence under control of a mini-calmodulin promoter ora mini-elongation factor 1 α (_(mini)EF1α) promoter.
 2. The nucleic acidconstruct of claim 1, wherein the viral vector is an adeno-associatedviral (AAV) vector.
 3. The nucleic acid construct of claim 1, whereinthe nucleic acid construct comprises a sequence selected from the groupconsisting of SEQ ID NO: 1 and SEQ ID NO:
 7. 4. A method for treating orpreventing a cholesterol storage disease or disorder in a subject, themethod comprising: administering a composition comprising the nucleicacid construct of claim 1 and a pharmaceutically acceptable viralcarrier to a subject, thereby treating or preventing the cholesterolstorage disease or disorder in the subject.
 5. The method of claim 4,wherein the cholesterol storage disease or disorder is Niemann-Pickdisease, Type C.
 6. (canceled)
 7. (canceled)
 8. The method of claim 4,wherein the subject is a mammal.
 9. The method of claim 4, wherein thenucleic acid construct is encapsidated with an AAV serotype 9 capsid.10. The method of claim 4, wherein the concentration of the nucleic acidconstruct in the composition is at least 5×10¹² gc/ml.
 11. The method ofclaim 4, wherein the pharmaceutically acceptable viral carrier is AAV.12. The method of claim 11, wherein the AAV is selected from the groupconsisting of AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh8,AAVrh10, AAVrh33, AAV rh34, AAV Anc80, or AAV PHP.B.
 13. The method ofclaim 4, wherein the pharmaceutically acceptable viral carrier comprisesa viral capsid selected from the group consisting of AAV2, AAV3, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh8, AAVrh10, AAVrh33, AAV rh34, AAVAnc80, or AAV PHP.B viral capsid.
 14. A method for treating Niemann-Pickdisease, Type C in a subject by gene therapy comprising administering acomposition comprising a therapeutically effective amount of a genetherapy construct comprising (1) a viral vector sequence; and (2) anNPC1 gene sequence under control of a mini-calmodulin promoter or a minielongation factor 1 α (_(mini)EF1α) promoter, and a pharmaceuticallyacceptable carrier.
 15. The method of claim 14, wherein the viral vectoris an adeno-associated viral (AAV) vector.
 16. The method of claim 14,wherein the gene therapy construct comprises a sequence selected fromthe group consisting of SEQ ID NO: 1 (AAV._(mini)CaMKII NPC1.RBG) andSEQ ID NO: 7 (pAAV-_(mini)EF1α-NPC1-RBG).
 17. (canceled)
 18. The methodof claim 14, wherein the subject is a mammal.
 19. The method of claim14, wherein the gene therapy construct is encapsidated with an AAVserotype 9 capsid.
 20. The method of claim 14, wherein the compositioncomprises the gene therapy construct at a concentration of 5×10¹² gc/mlor more.
 21. The method of claim 14, wherein the viral vector sequenceis AAV.
 22. The method of claim 21, wherein the AAV is selected from thegroup consisting of AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh33, AAV rh34, AAV Anc80, or AAV PHP.B.
 23. The methodof claim 14, wherein the gene therapy construct comprises a viral capsidselected from the group consisting of AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAV rh8, AAVrh10, AAVrh33, AAV rh34, AAV Anc80, or AAVPHP.B viral capsid.
 24. (canceled)
 25. (canceled)